Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma

ABSTRACT

Disclosed herein are therapeutic treatment protocols designed for the treatment of B cell lymphoma. These protocols are based upon therapeutic strategies which include the use of administration of immunologically active mouse/human chimeric anti-CD20 antibodies, radiolabeled anti-CD20 antibodies, and cooperative strategies comprising the use of chimeric anti-CD20 antibodies and radiolabeled anti-CD20 antibodies.

A. FIELD OF THE INVENTION

[0001] The references to be discussed throughout this document are setforth merely for the information described therein prior to the filingdates of this document, and nothing herein is to be construed as anadmission, either express or implied, that the references are “priorart” or that the inventors are not entitled to antedate suchdescriptions by virtue of prior inventions or priority based on earlierfiled applications.

[0002] The present invention is directed to the treatment of B celllymphoma using chimeric and radiolabeled antibodies to the B cellsurface antigen Bp35 (“CD20”).

B. BACKGROUND OF THE INVENTION

[0003] The immune system of vertebrates (for example, primates, whichinclude humans, apes, monkeys, etc.) consists of a number of organs andcell types which have evolved to: accurately and specifically recognizeforeign microorganisms (“antigen”) which invade the vertebrate-host;specifically bind to such foreign microorganisms; and, eliminate/destroysuch foreign microorganisms. Lymphocytes, amongst others, are criticalto the immune system. Lymphocytes are produced in the thymus, spleen andbone marrow (adult) and represent about 30% of the total white bloodcells present in the circulatory system of humans (adult). There are twomajor sub-populations of lymphocytes: T cells and B cells. T cells areresponsible for cell mediated immunity, while B cells are responsiblefor antibody production (humoral immunity). However, T cells and B cellscan be considered as interdependent—in a typical immune response, Tcells are activated when the T cell receptor binds to fragments of anantigen that are bound to major histocompatability complex (“MHC”)glycoproteins on the surface of an antigen presenting cell; suchactivation causes release of biological mediators (“interleukins”)which, in essence, stimulate B cells to differentiate and produceantibody (“immunoglobulins”) against the antigen.

[0004] Each B call within the host expresses a different antibody on itssurface—thus, one B cell will express antibody specific for one antigen,while another B cell will express antibody specific for a differentantigen. Accordingly, B cells are quite diverse, and this diversity iscritical to the immune system. In humans, each B cell can produce anenormous number of antibody molecules (ie about 10⁷ to 10⁸). Suchantibody production most typically ceases (or substantially decreases)when the foreign antigen has been neutralized. Occasionally, however,proliferation of a particular B cell will continue unabated; suchproliferation can result in a cancer referred to as “B cell lymphoma.”

[0005] T cells and B cells both comprise cell surface proteins which canbe utilized as “markers” for differentiation and identification. Onesuch human B cell marker is the human B lymphocyte-restricteddifferentiation antigen Bp35, referred to as “CD20.” CD20 is expressedduring early pre-B cell development and remains until plasma celldifferentiation. Specifically, the CD20 molecule may regulate a step inthe activation process which is required for cell cycle initiation anddifferentiation and is usually expressed at very high levels onneoplastic (“tumor”) B cells. CD20, by definition, is present on both“normal” B cells as well as “malignant” B cells, ie those B cells whoseunabated proliferation can lead to B cell lymphoma. Thus, the CD20surface antigen has the potential of serving as a candidate for“targeting” of B cell lymphomas.

[0006] In essence, such targeting can be generalized as follows:antibodies specific to the CD20 surface antigen of B cells are, eginjected into a patient. These anti-CD20 antibodies specifically bind tothe CD20 cell surface antigen of (ostensibly) both normal and malignantB cells; the anti-CD20 antibody bound to the CD20 surface antigen maylead to the destruction and depletion of neoplastic B cells.Additionally, chemical agents or radioactive labels having the potentialto destroy the tumor can be conjugated to the anti-CD20 antibody suchthat the agent is specifically “delivered” to, e.g., the neoplastic Bcells. Irrespective of the approach, a primary goal is to destroy thetumor: the specific approach can be determined by the particularanti-CD20 antibody which is utilized and, thus, the available approachesto targeting the CD20 antigen can vary considerably.

[0007] For example, attempts at such targeting of CD20 surface antigenhave been reported. Murine (mouse) monoclonal antibody 1F5 (an anti-CD20antibody) was reportedly administered by continuous intravenous infusionto B cell lymphoma patients. Extremely high levels (>2 grams) of 1F5were reportedly required to deplete circulating tumor cells, and theresults were described as being “transient.” Press et al., “MonoclonalAntibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas.” Blood69/2:584-591 (1987). A potential problem with this approach is thatnon-human monoclonal antibodies (eg, murine monoclonal antibodies)typically lack human effector functionality, ie they are unable to,inter alia, mediate complement dependent lysis or lyse human targetcells through antibody dependent cellular toxicity or Fc-receptormediated phagocytosis. Furthermore, non-human monoclonal antibodies canbe recognized by the human host as a foreign protein; therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody response, or “HAMA” response. Additionally, these“foreign” antibodies can be attacked by the immune system of the hostsuch that they are, in effect, neutralized before they reach theirtarget site.

[0008] Lymphocytes and lymphoma cells are inherently sensitive toradiotherapy for several reasons: the local emission of ionizingradiation of radiolabeled antibodies may kill cells with or without thetarget antigen (eg, CD20) in close proximity to antibody bound to theantigen; penetrating radiation may obviate the problem of limited accessto the antibody in bulky or poorly vascularized tumors; and, the totalamount of antibody required may be reduced. The radionuclide emitsradioactive particles which can damage cellular DNA to the point wherethe cellular repair mechanisms are unable to allow the cell to continueliving; therefore, if the target cells are tumors, the radioactive labelbeneficially kills the tumor cells. Radiolabeled antibodies, bydefinition, include the use of a radioactive substance which may requirethe need for precautions for both the patient (ie possible bone marrowtransplantation) as well as the health care provider (ie the need toexercise a high degree of caution when working with the radioactivity).

[0009] Therefore, an approach at improving the ability of murinemonoclonal antibodies to be effective in the treatment of B-celldisorders has been to conjugate a radioactive label or toxin to theantibody such that the label or toxin is localized at the tumor site.For example, the above-referenced IF5 antibody has been “labeled” withiodine-131 (“¹³¹I”) and was reportedly evaluated for biodistribution intwo patients. See Eary, J. F. et al., “Imaging and Treatment of B-CellLymphoma” J. Nuc. Med. 31/8:1257-1268 (1990); see also, Press, O. W. etal., “Treatment of Refractory Non-Hodgkin's Lymphoma with RadiolabeledMB-1 (Anti-CD37) Antibody” J. Clin. Onc. 7/8:1027-1038 (1989)(indication that one patient treated with ¹³¹I-labeled IF-5 achieved a“partial response”); Goldenberg, D. M. et al., “Targeting, Dosimetry andRadioimmunotherapy of B-Cell Lymphomas with Iodine-131-Labeled LL2Monoclonal Antibody” J. Clin. Onc. 9/4:548-564 (1991) (three of eightpatients receiving multiple injections reported to have developed a HAMAresponse); Appelbaum, F. R. “Radiolabeled Monoclonal Antibodies in theTreatment of Non-Hodgkin's Lymphoma” Hem./Onc. Clinics of N.A.5/5:1013-1025 (1991) (review article); Press, O. W. et al“Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Autologous BoneMarrow Support.” New England Journal of Medicine 329/17: 1219-12223(1993) (iodine-131 labeled anti-CD20 antibody IF5 and B1); and Kaminski,M. G. et al “Radioimmunotherapy of B-Cell Lymphoma with [¹³¹I ] Anti-B1(Anti-CD20) Antibody”. NEJM329/7 (1993) (iodine-131 labeled anti-CD20antibody B1; hereinafter “Kaminski”).

[0010] Toxins (ie chemotherapeutic agents such as doxorubicin ormitomycin C) have also been conjugated to antibodies. See, for example,PCT published application WO 92/07466 (published May 14, 1992).

[0011] “Chimeric” antibodies, ie antibodies which comprise portions fromtwo or more different species (eg, mouse and human) have been developedas an alternative to “conjugated” antibodies. For example, Liu, A. Y. etal., “Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20with Potent Fc-Dependent Biologic Activity” J. Immun. 139/10:3521-3526(1987), describes a mouse/human chimeric antibody directed against theCD20 antigen. See also, PCT Publication No. WO 88/04936. However, noinformation is provided as to the ability, efficacy or practicality ofusing such chimeric antibodies for the treatment of B cell disorders inthe reference. It is noted that in vitro functional assays (egcomplement dependent lysis (“CDC”); antibody dependent cellularcytotoxicity (“ADCC”), etc.) cannot inherently predict the in vivocapability of a chimeric antibody to destroy or deplete target cellsexpressing the specific antigen. See, for example, Robinson, R. D. etal., “Chimeric mouse-human anti-carcinoma antibodies that mediatedifferent anti-tumor cell biological activities,” Hum. Antibod.Hybridomas 2:84-93 (1991) (chimeric mouse-human antibody havingundetectable ADCC activity). Therefore, the potential therapeuticefficacy of chimeric antibody can only truly be assessed by in vivoexperimentation.

[0012] What is needed, and what would be a great advance in the art, aretherapeutic approaches targeting the CD20 antigen for the treatment of Bcell lymphomas in primates, including, but not limited to, humans.

C. SUMMARY OF THE INVENTION

[0013] Disclosed herein are therapeutic methods designed for thetreatment of B cell disorders, and in particular, B cell lymphomas.These protocols are based upon the administration of immunologicallyactive chimeric anti-CD20 antibodies for the depletion of peripheralblood B cells, including B cells associated with lymphoma;administration of radiolabeled anti-CD20 antibodies for targetinglocalized and peripheral B cell associated tumors; and administration ofchimeric anti-CD20 antibodies and radiolabeled anti-CD20 antibodies in acooperative therapeutic strategy.

D. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a diagrammatic representation of a tandem chimericantibody expression vector useful in the production of immunologicallyactive chimeric anti-CD20 antibodies (“TCAE 8”);

[0015]FIGS. 2A through 2E are the nucleic acid sequence of the vector ofFIG. 1;

[0016]FIGS. 3A through 3F are the nucleic acid sequence of the vector ofFIG. 1 further comprising murine light and heavy chain variable regions(“anti-CD20 in TCAE 8”);

[0017]FIG. 4 is the nucleic acid and amino acid sequences (including CDRand framework regions) of murine variable region light chain derivedfrom murine anti-CD20 monoclonal antibody 2B8;

[0018]FIG. 5 is the nucleic acid and amino acid sequences (including CDRand framework regions) of murine variable region heavy chain derivedfrom murine anti-CD20 monoclonal antibody 2B8;

[0019]FIG. 6 are flow cytometry results evidencing binding offluorescent-labeled human C1q to chimeric anti-CD20 antibody, including,as controls labeled C1q; labeled C1q and murine anti-CD20 monoclonalantibody 2B8; and labeled C1q and human IgGl,k;

[0020]FIG. 7 represents the results of complement related lysiscomparing chimeric anti-CD20 antibody and murine anti-CD20 monoclonalantibody 2B8;

[0021]FIG. 8 represents the results of antibody mediated cellularcytotoxicity with in vivo human effector cells comparing chimericanti-CD20 antibody and 2B8;

[0022]FIGS. 9A, 9B and 9C provide the results of non-human primateperipheral blood B lymphocyte depletion after infusion of 0.4 mg/kg (A);1.6 mg/kg (B); and 6.4 mg/kg (C) of immunologically active chimericanti-CD20 antibody;

[0023]FIG. 10 provides the results of, inter alia, non-human primateperipheral blood B lymphocyte depletion after infusion of 0.01 mg/kg ofimmunologically active chimeric anti-CD20 antibody;

[0024]FIG. 11 provides results of the tumoricidal impact of Y2B8 in amouse xenographic model utilizing a B cell lymphoblastic tumor;

[0025]FIG. 12 provides results of the tumoricidal impact of C2B8 in amouse xenographic model utilizing a B cell lymphoblastic tumor;

[0026]FIG. 13 provides results of the tumoricidal impact of acombination of Y2B8 and C2B8 in a mouse xenographic model utilizing a Bcell lymphoblastic tumor; and

[0027]FIGS. 14A and 14B provide results from a Phase I/II clinicalanalysis of C2B8 evidencing B-cell population depletion over time forpatients evidencing a partial remission of the disease (14A) and a minorremission of the disease (14B).

E. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0028] Generally, antibodies are composed of two light chains and twoheavy chain molecules; these chains form a general “Y” shape, with bothlight and heavy chains forming the arms of the Y and the heavy chainsforming the base of the Y. Light and heavy chains are divided intodomains of structural and functional homology. The variable domains ofboth the light (“V_(L)”) and the heavy (“V_(H)”) chains determinerecognition and specificity. The constant region domains of light(“C_(L)”) and heavy (“C_(H)”) chains confer important biologicalproperties, eg antibody chain association, secretion, transplacentalmobility, Fc receptor binding complement binding, etc. The series ofevents leading to immunoglobulin gene expression in the antibodyproducing cells are complex. The variable domain region gene sequencesare located in separate germ line gene segments referred to as “V_(H),”“D,” and “J_(H),” or “V_(L)” and “J_(L).” These gene segments are joinedby DNA rearrangements to form the complete V regions expressed in heavyand light chains, respectively. The rearranged, joined V segments(V_(L)-J_(L) and V_(H)-D-J_(H)) then encode the complete variableregions or antigen binding domains of light and heavy chains,respectively.

[0029] Serotherapy of human B cell lymphomas using an anti-CD20 murinemonoclonal antibody (1F5) has been described by Press et al., (69 Blood584, 1987, supra); the reported therapeutic responses, unfortunately,were transient. Additionally, 25% of the tested patients reportedlydeveloped a human anti-mouse antibody (HAMA) response to theserotherapy. Press et al., suggest that these antibodies, conjugated totoxins or radioisotopes, might afford a more lasting clinical benefitthan the unconjugated antibody.

[0030] Owing to the debilitating effects of B cell lymphoma and the veryreal need to provide viable treatment approaches to this disease, wehave embarked upon different approaches having a particular antibody,2B8, as the common link between the approaches. One such approachadvantageously exploits the ability of mammalian systems to readily andefficiently recover peripheral blood B cells; using this approach, weseek to, in essence, purge or deplete B cells in peripheral blood andlymphatic tissue as a means of also removing B cell lymphomas. Weaccomplish this by utilization of, inter alia, immunologically active,chimeric anti-CD20 antibodies. In another approach, we seek to targettumor cells for destruction with radioactive labels.

[0031] As used herein, the term “anti-CD20 antibody” is an antibodywhich specifically recognizes a cell surface non-glycosylatedphosphoprotein of 35,000 Daltons, typically designated as the human Blymphocyte restricted differentiation antigen Bp35, commonly referred toas CD20. As used herein, the term “chimeric” when used in reference toanti-CD20 antibodies, encompasses antibodies which are most preferablyderived using recombinant deoxyribonucleic acid techniques and whichcomprise both human (including immunologically “related” species, eg,chimpanzee) and non-human components: the constant region of thechimeric antibody is most preferably substantially identical to theconstant region of a natural human antibody; the variable region of thechimeric antibody is most preferably derived from a non-human source andhas the desired antigenic and specificity to the CD20 cell surfaceantigen. The non-human source can be any vertebrate source which can beused to generate antibodies to a human CD20 cell surface antigen ormaterial comprising a human CD20 cell surface antigen. Such non-humansource includes, but is not limited to, rodents (eg, rabbit, rat, mouse,etc.) and non-human primates (eg, Old World Monkey, Ape, etc.). Mostpreferably, the non-human component (variable region) is derived from amurine source. As used herein, the phrase “immunologically active” whenused in reference to chimeric anti-CD20 antibodies, means a chimericantibody which binds human C1q, mediates complement dependent lysis(“CDC”) of human B lymphoid cell lines, and lyses human target cellsthrough antibody dependent cellular cytotoxicity (“ADCC”). As usedherein, the phrases “indirect labeling” and “indirect labeling approach”both mean that a chelating agent is covalently attached to an antibodyand at least one radionuclide is inserted into the chelating agent.Preferred chelating agents and radionuclides are set forth inSrivagtava, S. C. and Mease, R. C.,“Progress in Research on Ligands,Nuclides and Techniques for Labeling Monoclonal Antibodies,” Nucl. Med.Bio. 18/6: 589-603 (1991) (“Srivagtava”) which is incorporated herein byreference. A particularly preferred chelating agent is1-isothiocycmatobenzyl-3-methyldiothelene triaminepent acetic acid(“MX-DTPA”); particularly preferred radionuclides for indirect labelinginclude indium [111] and yttrium [90]. As used herein, the phrases“direct labeling” and “direct labeling approach” both mean that aradionuclide is covalently attached directly to an antibody (typicallyvia an amino acid residue). Preferred radionuclides are provided inSrivagtava; a particularly preferred radionuclide for direct labeling isiodine [131] covalently attached via tyrosine residues. The indirectlabeling approach is particularly preferred.

[0032] The therapeutic approaches disclosed herein are based upon theability of the immune system of primates to rapidly recover, orrejuvenate, peripheral blood B cells. Additionally, because theprincipal immune response of primates is occasioned by T cells, when theimmune system has a peripheral blood B cell deficiency, the need for“extraordinary” precautions (ie patient isolation, etc.) is notnecessary. As a result of these and other nuances of the immune systemsof primates, our therapeutic approach to B cell disorders allows for thepurging of peripheral blood B cells using immunologically activechimeric anti-CD20 antibodies.

[0033] Because peripheral blood B cell disorders, by definition, canindicate a necessity for access to the blood for treatment, the route ofadministration of the immunologically active chimeric anti-CD20antibodies and radioalabeled anti-CD20 antibodies is preferablyparenteral; as used herein, the term “parenteral” includes intravenous,intramuscular, subcutaneous, rectal, vaginal or intraperitonealadministration. Of these, intravenous administration is most preferred.

[0034] The immunologically active chimeric anti-CD20 antibodies andradiolabeled anti-CD20 antibodies will typically be provided by standardtechnique within a pharmaceutically acceptable buffer, for example,sterile saline, sterile buffered water, propylene glycol, combinationsof the foregoing, etc. Methods for preparing parenteraly administerableagents are described in Pharmaceutical Carriers & Formulations, Martin,Remington's Pharmaceutical Sciences, 15th Ed. (Mack Pub. Co., Easton,Pa. 1975), which is incorporated herein by reference.

[0035] The specific, therapeutically effective amount of immunologicallyactive chimeric anti-CD20 antibodies useful to produce a uniquetherapeutic effect in any given patient can be determined by standardtechniques well known to those of ordinary skill in the art.

[0036] Effective dosages (ie therapeutically effective amounts) of theimmunologically active chimeric anti-CD20 antibodies range from about0.001 to about 30 mg/kg body weight, more preferably from about 0.01 toabout 25 mg/kg body weight, and most preferably from about 0.4 to about20.0 mg/kg body weight. Other dosages are viable; factors influencingdosage include, but are not limited to, the severity of the disease;previous treatment approaches; overall health of the patient; otherdiseases present, etc. The skilled artisan is readily credited withassessing a particular patient and determining a suitable dosage thatfalls within the ranges, or if necessary, outside of the ranges.

[0037] Introduction of the immunologically active chimeric anti-CD20antibodies in these dose ranges can be carried out as a single treatmentor over a series of treatments. With respect to chimeric antibodies, itis preferred that such introduction be carried out over a series oftreatments; this preferred approach is predicated upon the treatmentmethodology associated with this disease. While not wishing to be boundby any particular theory, because the immunologically active chimericanti-CD20 antibodies are both immunologically active and bind to CD20,upon initial introduction of the immunologically active chimericanti-CD20 antibodies to the individual, peripheral blood B celldepletion will begin; we have observed a nearly complete depletionwithin about 24 hours post treatment infusion. Because of this,subsequent introduction(s) of the immunologically active chimericanti-CD20 antibodies (or radiolabeled anti-CD20 antibodies) to thepatient is presumed to: a) clear remaining peripheral blood B cells; b)begin B cell depletion from lymph nodes; c) begin B cell depletion fromother tissue sources, eg, bone marrow, tumor, etc. Stated again, byusing repeated introductions of the immunologically active chimericanti-CD20 antibodies, a series of events take place, each event beingviewed by us as important to effective treatment of the disease. Thefirst “event” then, can be viewed as principally directed tosubstantially depleting the patient's peripheral blood B cells; thesubsequent “events” can be viewed as either principally directed tosimultaneously or serially clearing remaining B cells from the systemclearing lymph node B cells, or clearing other tissue B cells.

[0038] In effect, while a single dosage provides benefits and can beeffectively utilized for disease treatment/management, a preferredtreatment course can occur over several stages; most preferably, betweenabout 0.4 and about 20 mg/kg body weight of the immunologically activechimeric anti-CD20 antibodies is introduced to the patient once a weekfor between about 2 to 10 weeks, most preferably for about 4 weeks.

[0039] With reference to the use of radiolabeled anti-CD20 antibodies, apreference is that the antibody is non-chimeric; this preference ispredicted upon the significantly longer circulating half-life ofchimeric antibodies vis-a-vis murine antibodies (ie with a longercirculating half-life, the radionuclide is present in the patient forextended periods). However, radiolabeled chimeric antibodies can bebeneficially utilized with lower milli-Curries (“mCi”) dosages used inconjunction with the chimeric antibody relative to the murine antibody.This scenario allows for a decrease in bone marrow toxicity to anacceptable level, while maintaining therapeutic utility.

[0040] A variety of radionuclides are applicable to the presentinvention and those skilled in the art are credited with the ability toreadily determine which radionuclide is most appropriate under a varietyof circumstances. For example, iodine [131] is a well known radionuclideused for targeted immunotherapy. However, the clinical usefulness ofiodine [131] can be limited by several factors including: eight-dayphysical half-life; dehalogenation of iodinated antibody both in theblood and at tumor sites; and emission characteristics (eg large gammacomponent) which can be suboptimal for localized dose deposition intumor. With the advent of superior chelating agents, the opportunity forattaching metal chelating groups to proteins has increased theopportunities to utilize other radionuclides such as indium [131] andyttrium [90]. Yttrium [90] provides several benefits for utilization inradioimmunotherapeutic applications: the 64 hour half-life of yttrium[90] is long enough to allow antibody accumulation by tumor and, unlikeeg iodine [131], yttrium [90] is a pure beta emitter of high energy withno accompanying gamma irradiation in its decay, with a range in tissueof 100 to 1000 cell diameters. Furthermore, the minimal amount ofpenetrating radiation allows for outpatient administration of yttrium[90]-labeled antibodies. Furthermore, interalization of labeled antibodyis not required for cell killing, and the local emission of ionizingradiation should be lethal for adjacent tumor cells lacking the targetantigen.

[0041] One non-therapeutic limitation to yttrium [90] is based upon theabsence of significant gamma radiation making imaging therewithdifficult. To avoid this problem, a diagnostic “imaging” radionuclide,such as indium [111], can be utilized for determining the location andrelative size of a tumor prior to the administration of therapeutic doesof yttrium [90]-labeled anti-CD20. Indium [111] is particularlypreferred as the diagnostic radionuclide because: between about 1 toabout 10 mCi can be safely administered without detectable toxicity; andthe imaging data is generally predictive of subsequent yttrium[90]-labeled antibody distribution. Most imaging studies utilize 5mCiindium [111]-labeled antibody because this dose is both safe and hasincreased imaging efficiency compared with lower doses, with optimalimaging occurring at three to six days after antibody administration.See, for example, Murray J. L., 26 J. Nuc. Med. 3328 (1985) andCarraguillo, J. A. et al, 26 J. Nuc. Med. 67 (1985).

[0042] Effective single treatment dosages (ie therapeutically effectiveamounts) of yttrium [90] labeled anti-CD20 antibodies range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of iodine[131] labeled anti-CD20 antibodies range from between about 5 and about70 mCi, more preferably between about 5 and about 40 mCi. Effectivesingle treatment ablative dosages (ie may require autologous bone marrowtransplantation) of iodine [131] labeled anti-CD20 antibodies range frombetween about 30 and about 600 mCi, more preferably between about 50 andless than about 500 mCi. In conjunction with a chimeric anti-CD20antibody, owing to the longer circulating half life vis-a-vis murineantibodies, an effective single treatment non-marrow ablative dosages ofiodine [131] labeled chimeric anti-CD20 antibodies range from betweenabout 5 and about 40 mCi, more preferably less than about 30 mCi.Imaging criteria for, eg the indium [111] label, are typically less thanabout 5 mCi.

[0043] With respect to radiolabeled anti-CD20 antibodies, therapytherewith can also occur using a single therapy treatment or usingmultiple treatments. Because of the radionuclide component, it ispreferred that prior to treatment, peripheral stem cells (“PSC”) or bonemarrow (“BM”) be “harvested” for patients experiencing potentially fatalbone marrow toxicity resulting from radiation. BM and/or PSC areharvested using standard techniques, and then purged and frozen forpossible reinfusion. Additionally, it is most preferred that prior totreatment a diagnostic dosimetry study using a diagnostic labeledantibody (eg using indium [111]) be conducted on the patient, a purposeof which is to ensure that the therapeutically labeled antibody (egusing yttrium [90]) will not become unnecessarily “concentrated” in anynormal organ or tissue.

[0044] Chimeric mouse/human antibodies have been described. See, forexample, Morrison, S. L. et al., PNAS I1:6851-6854 (November 1984);European Patent Publication No. 173494; Boulianne, G. L. et al., Nature312:642 (December 1984); Neubeiger, M. S. et al., Nature 314:268 (March1985); European Patent Publication No. 125023; Tan et al., J. Immunol.135:8564 (November 1985); Sun, L. K. et al., Hybridoma 5/1:517 (1986);Sahagan et al., J. Immunol. 137:1066-1074 (1986). See generally, Muron,Nature 312:597 (December 1984); Dickson, Genetic Engineering News 5 /3(March 1985); Marx, Science 229 455 (August 1985); and Morrison Science229:1202-1207 (September 1985). Robinson et al., in PCT PublicationNumber WO 88/04936 describe a chimeric antibody with human constantregion and murine variable region, having specificity to an epitope ofCD20; the murine portion of the chimeric antibody of the Robinsonreferences is derived from the 2H7 mouse monoclonal antibody (gamma 2 b,kappa). While the reference notes that the described chimeric antibodyis a “prime candidate” for the treatment of B cell disorders, thisstatement can be viewed as no more than a suggestion to those in the artto determine whether or not this suggestion is accurate for thisparticular antibody, particularly because the reference lacks any datato support an assertion of therapeutic effectiveness, and importantly,data using higher order mammals such as primates or humans.

[0045] Methodologies for generating chimeric antibodies are available tothose in the art. For example, the light and heavy chains can beexpressed separately, using, for example, immunoglobulin light chain andimmunoglobulin heavy chains in separate plasmids. These can then bepurified and assembled in vitro into complete antibodies; methodologiesfor accomplishing such assembly have been described. See, for example,Scharff, M., Harvey Lectures 69:125 (1974). In vitro reaction parametersfor the formation of IgG antibodies from reduced isolated light andheavy chains have also been described. See, for example, Beychok, S.,Cells of Immunoglobulin Synthesis, Academic Press, New York, p. 69,1979. Co-expression of light and heavy chains in the same cells toachieve intracellular association and linkage of heavy and light chainsinto complete H₂L₂ IgG antibodies is also possible. Such co-expressioncan be accomplished using either the same or different plasmids in thesame host cell.

[0046] Another approach, and one which is our most preferred approachfor developing a chimeric non-human/human anti-CD20 antibody, is basedupon utilization of an expression vector which includes, ab initio, DNAencoding heavy and light chain constant regions from a human source.Such a vector allows for inserting DNA encoding non-human variableregion such that a variety of non-human anti-CD20 antibodies can begenerated, screened and analyzed for various characteristics (eg type ofbinding specificity, epitope binding regions, etc.); thereafter, cDNAencoding the light and heavy chain variable regions from a preferred ordesired anti-CD20 antibody can be incorporated into the vector. We referto these types of vectors as Tandem Chimeric Antibody Expression(“TCAE”) vectors. A most preferred TCAE vector which was used togenerate immunologically active chimeric anti-CD20 antibodies fortherapeutic treatment of lymphomas is TCAE 8. TCAE 8 is a derivative ofa vector owned by the assignee of this patent document, referred to asTCAE 5.2 the difference being that in TCAE 5.2, the translationinitiation start site of the dominant selectable marker (neomycinphosphostransferase, “NEO”) is a consensus Kozak sequence, while forTCAE 8, this region is a partially impaired consensus Kozak sequence.Details regarding the impact of the initiation start site of thedominant selectable marker of the TCAE vectors (also referred to as“ANEX vector”) vis-a-vis protein expression are disclosed in detail inthe co-pending application filed herewith.

[0047] TCAE 8 comprises four (4) transcriptional cassettes, and theseare in tandem order, ie a human immunoglobulin light chain absent avariable region; a human immunoglobulin heavy chain absent a variableregion; DHFR; and NEO. Each transcriptional cassette contains its owneukaryotic promoter and polyadenylation region (reference is made toFIG. 1 which is a diagrammatic representation of the TCAE 8 vector).Specifically:

[0048] 1) the CMV promoter/enhancer in front of the immunoglobulin heavychain is a truncated version of the promoter/enhancer in front of thelight chain, from the Nhe I site at −350 to the Sst I site at −16 (see,41 Cell 521, 1985).

[0049] 2) a human immunoglobulin light chain constant region was derivedvia amplification of cDNA by a PCR reaction. In TCAE 8, this was thehuman immunoglobulin light chain kappa constant region (Kabat numbering,amino acids 108-214, allotype Km 3, (see, Kabat, E. A. “Sequences ofproteins of immunological interest,” NIH Publication, Fifth Ed. No.91-3242, 1991)), and the human immunoglobulin heavy chain gamma 1constant region (Kabat numbering amino acids 114-478, allotype Gmla,Gmlz). The light chain was isolated from normal human blood (IDECPharmaceuticals Corporation, La Jolla, Calif.); RNA therefrom was usedto synthesize cDNA which was then amplified using PCR techniques(primers were derived vis-a-vis the consensus from Kabat). The heavychain was isolated (using PCR techniques) from cDNA prepared from RNAwhich was in turn derived from cells transfected with a human IgG1vector (see, 3 Prot. Eng. 531, 1990; vector pN_(γ1)62). Two amino acidswere changed in the isolated human IgG1 to match the consensus aminoacid sequence from Kabat, to wit: amino acid 225 was changed from valineto alanine (GTT to GCA), and amino acid 287 was changed from methionineto lysine (ATG to AAG);

[0050] 3) The human immunoglobulin light and heavy chain cassettescontain synthetic signal sequences for secretion of the immunoglobulinchains;

[0051] 4) The human immunoglobulin light and heavy chain cassettescontain specific DNA restriction sites which allow for insertion oflight and heavy immunoglobulin variable regions which maintain thetransitional reading frame and do not alter the amino acids normallyfound in immunoglobulin chains;

[0052] 5) The DHFR cassette contained its own eukaryotic promoter (mousebeta globin major promoter, “BETA”) and polyadenylation region (bovinegrowth hormone polyadenylation, “BGH”); and

[0053] 6) The NEO cassette contained its own eukaryotic promoter (BETA)and polyadenylation region (SV40 early polyadenylation, “SV”).

[0054] With respect to the TCAE 8 vector and the NEO cassette, the Kozakregion was a partially impaired consensus Kozak sequence (which includedan upstream Cla I site):            ClaI    −3      +1 GGGAGCTTGG ATCCATccTct ATG Gtt

[0055] (In the TCAE 5.2 vector, the change is between the ClaI and ATGregions, to wit: ccAcc.)

[0056] The complete sequence listing of TCAE 8 (including the specificcomponents of the four transcriptional cassettes) is set forth in FIG. 2(SEQ. ID. NO. 1).

[0057] As will be appreciated by those in the art, the TCAE vectorsbeneficially allow for substantially reducing the time in generating theimmunologically active chimeric anti-CD20 antibodies. Generation andisolation of non-human light and heavy chain variable regions, followedby incorporation thereof within the human light chain constanttranscriptional cassette and human heavy chain constant transcriptionalcassette, allows for production of immunologically active chimericanti-CD20 antibodies.

[0058] We have derived a most preferred non-human variable region withspecificity to the CD20 antigen using a murine source and hybridomatechnology. Using polymerase chain reaction (“PCR”) techniques, themurine light and heavy variable regions were cloned directly into theTCAE 8 vector—this is the most preferred route for incorporation of thenon-human variable region into the TCAE vector. This preference isprincipally predicated upon the efficiency of the PCR reaction and theaccuracy of insertion. However, other equivalent procedures foraccomplishing this task are available. For example, using TCAE 8 (or anequivalent vector), the sequence of the variable region of a non-humananti-CD20 antibody can be obtained, followed by oligonucleotidesynthesis of portions of the sequence or, if appropriate, the entiresequence; thereafter, the portions or the entire synthetic sequence canbe inserted into the appropriate locations within the vector. Thoseskilled in the art are credited with the ability to accomplish thistask.

[0059] Our most preferred immunologically active chimeric anti-CD20antibodies were derived from utilization of TCAE 8 vector which includedmurine variable regions derived from monoclonal antibody to CD20; thisantibody (to be discussed in detail, infra), is referred to as “2B8.”The complete sequence of the variable regions obtained from 2B8 in TCAE8 (“anti-CD20 in TCAE 8”) is set forth in FIG. 3 (SEQ. ID. NO. 2).

[0060] The host cell line utilized for protein expression is mostpreferably of mammalian origin; those skilled in the art are creditedwith ability to preferentially determine particular host cell lineswhich are best suited for the desired gene product to be expressedtherein. Exemplary host cell lines include, but are not limited to, DG44and DUXBll (Chinese Hamster Ovary lines, DHFR minus), HELA (humancervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVIwith SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3(mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma),P3×63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells),RAJI (human lymphocyte) and 293 (human kidney). Host cell lines aretypically available from commercial services, the American TissueCulture Collection or from published literature.

[0061] Preferably the host cell line is either DG44 (“CHO”) or SP2/O.See Urland, G. et al., “Effect of gamma rays and the dihydrofolatereductase locus: deletions and inversions.” Som. Cell & Mol. Gen.12/6:555-566 (1986), and Shulman, M. et al., “A better cell line formaking hybridomas secreting specific antibodies.” Nature 276:269 (1978),respectively. Most preferably, the host cell line is DG44. Transfectionof the plasmid into the host cell can be accomplished by any techniqueavailable to those in the art. These include, but are not limited to,transfection (including electrophoresis and electroporation), cellfusion with enveloped DNA, microinjection, and infection with intactvirus. See, Ridgway, A. A. G. “Mammalian Expression Vectors.” Chapter24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths,Boston, Mass. 1988). Most preferably, plasmid introduction into the hostis via electroporation.

F. EXAMPLES

[0062] The following examples are not intended, nor are they to beconstrued, as limiting the invention. The examples are intended toevidence: dose-imaging using a radiolabeled anti-CD20 antibody (“I2B8”);radiolabeled anti-CD20 antibody (“Y2B8”); and immunologically active,chimeric anti-CD20 antibody (“C2B8”) derived utilizing a specific vector(“TCAE 8”) and variable regions derived from murine anti-CD20 monoclonalantibody (“2B8”).

[0063] I. RADIOLABELED ANTI-CD20 ANTIBODY 2B8

[0064] A. Anti-CD20 Monoclonal Antibody (Murine) Production (“2B8”)

[0065] BALB/C mice were repeatedly immunized with the humanlymphoblastoid cell line SB (see, Adams, R. A. et al., “Directimplantation and serial transplantation of human acute lymphoblasticleukemia in hamsters, SB-2.” Can Res 28:1121-1125 (1968); this cell lineis available from the American Tissue Culture Collection, Rockville,Md., under ATCC accession number ATCC CCL 120), with weekly injectionsover a period of 3-4 months. Mice evidencing high serum titers ofanti-CD20 antibodies, as determined by inhibition of known CD20-specificantibodies (anti-CD20 antibodies utilized were Leu 16, BecktonDickinson, San Jose, Calif., Cat. No. 7670; and Bl, Coulter Corp.,Hialeah, Fla., Cat. No. 6602201) were identified; the spleens of suchmice were then removed. Spleen cells were fused with the mouse myelomaSP2/0 in accordance with the protocol described in Einfeld, D. A. etal., (1988) EMBO 7:711 (SP2/0 has ATCC accession no. ATCC CRL 8006).

[0066] Assays for CD20 specificity were accomplished byradioimmunoassay. Briefly, purified anti-CD20 Bl was radiolabeled withI¹²⁵ by the iodobead method as described in Valentine, M. A. et al.,(1989) J. Biol. Chem. 264:11282. (I¹²⁵ Sodium Iodide, ICN, Irvine,Calif., Cat. No. 28665H). Hybridomas were screened by co-incubation of0.05 ml of media from each of the fusion wells together with 0.05 ml ofI¹²⁵ labeled anti-CD20 Bl (10 ng) in 1% BSA, PBS (pH 7.4), and 0.5 ml ofthe same buffer containing 100,000 SB cells. After incubation for 1 hrat room temperature, the cells were harvested by transferring to 96 welltiter plates (V&P Scientific, San Diego, Calif.), and washed thoroughly.Duplicate wells containing unlabeled anti-CD20 Bl and wells containingno inhibiting antibody were used as positive and negative controls,respectively. Wells containing greater than 50% inhibition were expandedand cloned. The antibody demonstrating the highest inhibition wasderived from the cloned cell line designated herein as “2B8.”

[0067] B. Preparation of2B8-MX-DTPA Conjugate

[0068] i. MX-DTPA

[0069] Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (“carbon-14 labeled MX-DTPA”) was used as achelating agent for conjugation of radiolabel to 2B8. Manipulations ofMX-DTPA were conducted to maintain metal-free conditions, ie metal-freereagents were utilized and, when possible, polypropylene plasticcontainers (flasks, beakers, graduated cylinders, pipette tips) washedwith Alconox and rinsed with Milli-Q water, were similarly utilized.MX-DTPA was obtained as a dry solid from Dr. Otto Gansow (NationalInstitute of Health, Bethesda, Md.) and stored desiccated at 4° C.(protected from light), with stock solutions being prepared in Milli-Qwater at a concentration of 2-5 mM, with storage at −70° C. MX-DTPA wasalso obtained from Coulter Immunology (Hialeah, Florida) as the disodiumsalt in water and stored at −70° C.

[0070] ii. Preparation of 2B8

[0071] Purified 2B8 was prepared for conjugation with MX-DTPA bytransferring the antibody into metal-free 50 mM bicine-NaOff, pH 8.6,containing 150 mM NaCl, using repetitive buffer exchange with CENTRICON30™ spin filters (30,000D, MWCO; Amicon). Generally, 50-200 μL ofprotein (10 mg/nl) was added to the filter unit, followed by 2 mL ofbicine buffer. The filter was centrifuged at 4° C. in a Sorval SS-34rotor (6,000 rpm, 45 min.). Retentate volume was approximately 50-100μL; this process was repeated twice using the same filter. Retentate wastransferred to a polypropylene 1.5 mL screw cap tube, assayed forprotein, diluted to 10.0 mg/mL and stored at 4° C. until utilized;protein was similarly transferred into 50 mM sodium citrate, pH 5.5,containing 150 mM NaCl and 0.05% sodium azide, using the foregoingprotocol.

[0072] iii. Conjugation of 2B8 with MX-DTPA

[0073] Conjugation of 2B8 with MX-DTPA was performed in polypropylenetubes at ambient temperature. Frozen MX-DTPA stock solutions were thawedimmediately prior to use. 50-200 mL of protein at 10 mg/mL were reactedwith MX-DTPA at a molar ratio of MX-DTPA-to-2B8 of 4:1. Reactions wereinitiated by adding the MX-DTPA stock solution and gently mixing; theconjugation was allowed to proceed overnight (14 to 20 hr), at ambienttemperature. Unreacted MX-DTPA was removed from the conjugate bydialysis or repetitive ultrafiltration, as described above in ExampleI.B.ii, into metal-free normal saline (0.9% w/v) containing 0.05% sodiumazide. The protein concentration was adjusted to 10 mg/mL and stored at4° C. in a polypropylene tube until radiolabeled.

[0074] iv. Determination of MX-DTPA Incorporation

[0075] MX-DTPA incorporation was determined by scintillation countingand comparing the value obtained with the purified conjugate to thespecific activity of the carbon-[14]-labeled MX-DTPA. For certainstudies, in which non-radioactive MX-DTPA (Coulter Immunology) wasutilized, MX-DTPA incorporation was assessed by incubating the conjugatewith an excess of a radioactive carrier solution of yttrium-[90] ofknown concentration and specific activity.

[0076] A stock solution of yttrium chloride of known concentration wasprepared in metal-free 0.05 N HCl to which carrier-free yttrium-[90](chloride salt) was added. An aliquot of this solution was analyzed byliquid scintillation counting to determine an accurate specific activityfor this reagent. A volume of the yttrium chloride reagent equal to3-times the number of mols of chelate expected to be attached to theantibody, (typically 2 mol/mol antibody), was added to a polypropylenetube, and the pH adjusted to 4.0-4.5 with 2 M sodium acetate. Conjugatedantibody was subsequently added and the mixture incubated 15-30 min. atambient temperature. The reaction was quenched by adding 20 mM EDTA to afinal concentration of 1 mM and the pH of the solution adjusted toapproximately pH 6 with 2M sodium acetate.

[0077] After a 5 min. incubation, the entire volume was purified byhigh-performance, size-exclusion chromatography (described infra). Theeluted protein-containing fractions were combined, the proteinconcentration determined, and an aliquot assayed for radioactivity. Thechelate incorporation was calculated using the specific activity of theyttrium-[90] chloride preparation and the protein concentration.

[0078] v. Immunoreactivity of 2B8-MX-DTPA

[0079] The immunoreactivity of conjugated 2B8 was assessed usingwhole-cell ELISA. Mid-log phase SB cells were harvested from culture bycentrifugation and washed two times with 1×HBSS. Cells were diluted to1-2×10⁶ cells/mL in HBSS and aliquoted into 96-well polystyrenemicrotiter plates at 50,000-100,000 cells/well. The plates were driedunder vacuum for 2 h. at 40-45° C. to fix the cells to the plastic;plates were stored dry at −20° C. until utilized. For assay, the plateswere warmed to ambient temperature immediately before use, then blockedwith 1×PBS, pH 7.2-7.4 containing 1% BSA (2 h). Samples for assay werediluted in 1×PBS/1% BSA, applied to plates and serially diluted (1:2)into the same buffer. After incubating plates for 1 h. at ambienttemperature, the plates were washed three times with 1×PBS. Secondaryantibody (goat anti-mouse IgG1-specific HRP conjugate 50 μL) was addedto wells (1:1500 dilution in 1×PBS/1% BSA) and incubated 1 h. at ambienttemperature. Plates were washed four times with 1×PBS followed by theaddition of ABTS substrate solution (50 mM sodium citrate, pH 4.5containing 0.01% ATBS and 0.001% H₂O₂). Plates were read at 405 nm after15-30 min. incubation. Antigen-negative HSB cells were included inassays to monitor non-specific binding. Immunoreactivity of theconjugate was calculated by plotting the absorbance values vs. therespective dilution factor and comparing these to values obtained usingnative antibody (representing 100% immunoreactivity) tested on the sameplate; several values on the linear portion of the titration profilewere compared and a mean value determined (data not shown).

[0080] vi. Preparation of Indium-[111]-Labeled 2B8-MX-DTPA (“I2B8”)

[0081] Conjugates were radiolabeled with carrier-free indium-[111]. Analiquot of isotope (0.1-2 mCi/mg antibody) in 0.05 M HCL was transferredto a polypropylene tube and approximately one-tenth volume of metal-free2 M HCl added. After incubation for 5 min., metal-free 2 M sodiumacetate was added to adjust the solution to pH 4.0-4.4. Approximately0.5 mg of 2B8-MX-DTPA was added from a stock solution of 10.0 mg/mL DTPAin normal saline, or 50 mM sodium citrate/150 mM NaCl containing 0.05%sodium azide, and the solution gently mixed immediately. The pH solutionwas checked with pH paper to verify a value of 4.0-4.5 and the mixtureincubated at ambient temperature for 15-30 min. Subsequently, thereaction was quenched by adding 20 mM EDTA to a final concentration of 1mM and the reaction mixture was adjusted to approximately pH 6.0 using 2M sodium acetate.

[0082] After a 5-10 min. incubation, uncomplexed radioisotope wasremoved by size-exclusion chromatography. The HPLC unit consisted ofWaters Model 6000 or TosoHaas Model TSK-6110 solvent delivery systemfitted, respectively, with a Waters U6K or Rheodyne 700 injection valve.Chromatographic separations were performed using a gel permeation column(BioRad SEC-250; 7.5×300 mm or comparable TosoHaas column) and a SEC-250guard column (7.5×100 mm). The system was equipped with a fractioncollector (Pharmacia Frac200) and a UV monitor fitted with a 280 nmfilter (Pharmacia model TV-1). Samples were applied and elutedisocratically using 1×PBS, pH 7.4, at 1.0 mL/min flow rate. One-halfmilliliter fractions were collected in glass tubes and aliquots of thesecounted in a gamma counter. The lower and upper windows were set to 100and 500 KeV respectively.

[0083] The radioincorporation was calculated by summing theradioactivity associated with the eluted protein peak and dividing thisnumber by the total radioactivity eluted from the column; this value wasthen expressed as a percentage (data not shown). In some cases, theradioincorporation was determined using instant thin-layerchromatography (“ITLC”). Radiolabeled conjugate was diluted 1:10 or 1:20in 1×PBS containing or 1×PBS/1 mM DTPA, then 1 μL was spotted 1.5 cmfrom one end of a 1×5 cm strip of ITLC SG paper. The paper was developedby ascending chromatography using 10% ammonium acetate in methanol:water(1:1;v/v). The strip was dried, cut in half crosswise, and theradioactivity associated with each section determined by gamma counting.The radioactivity associated with the bottom half of the strip(protein-associated radioactivity) was expressed as a percentage of thetotal radioactivity, determined by summing the values for both top andbottom halves (data not shown).

[0084] Specific activities were determined by measuring theradioactivity of an appropriate aliquot of the radiolabeled conjugate.This value was corrected for the counter efficiency (typically 75%) andrelated to the protein concentration of the conjugate, previouslydetermined by absorbance at 280 nm, and the resulting value expressed asmCi/mg protein.

[0085] For some experiments, 2B8-MX-DTPA was radiolabeled with indium[111] following a protocol similar to the one described above butwithout purification by HPLC; this was referred to as the“mix-and-shoot” protocol.

[0086] vii. Preparation of Yttrium-[90]-Labeled 2B8-MX-DTPA (“Y2B8”)

[0087] The same protocol described for the preparation of I2B8 wasfollowed for the preparation of the yttrium-[90]-labeled 2B8-MX-DTPA(“Y2B8”) conjugate except that 2 ng HCl was not utilized; allpreparations of yttrium-labeled conjugates were purified bysize-exclusion chromatography as described above.

[0088] C. Non-Human Animal Studies.

[0089] i. Biodistribution of Radiolabeled 2B8-MX-DTPA

[0090] I2B8 was evaluated for tissue biodistribution in six-to-eightweek old BALB/c mice. The radiolabeled conjugate was prepared usingclinical-grade 2B8-MX-DTPA following the “mix and shoot” protocoldescribed above. The specific activity of the conjugate was 2.3 mCi/mgand the conjugate was formulated in PBS, pH 7.4 containing 50mg/mL HSA.Mice were injected intravenously with 100 μL of I2B8 (approximately 21μCi) and groups of three mice were sacrificed by cervical dislocation at0, 24, 48, and 72 hours. After sacrifice, the tail, heart, lungs, liver,kidney, spleen, muscle, and femur were removed, washed and weighed; asample of blood was also removed for analysis. Radioactivity associatedwith each specimen was determined by gamma counting and the presentinjected dose per gram tissue subsequently determined. No attempt wasmade to discount the activity contribution represented by the bloodassociated with individual organs.

[0091] In a separate protocol, aliquots of 2B8-MX-DTPA incubated at 4°C. and 30° C. for 10 weeks were radiolabeled with indium-[111] to aspecific activity of 2.1 mCi/mg for both preparations. These conjugateswere then used in biodistribution studies in mice as described above.

[0092] For dosimetry determinations, 2B8-MX-DTPA was radiolabeled withindium-[111] to a specific activity of 2.3 mCi/mg and approximately 1.1μCi was injected into each of 20 BALB/c mice. Subsequently, groups offive mice each were sacrificed at 1, 24, 48 and 72 hours and theirorgans removed and prepared for analysis. In addition, portions of theskin, muscle and bone were removed and processed for analysis; the urineand feces were also collected and analyzed for the 24-72 hour timepoints.

[0093] Using a similar approach, 2B8-MX-DTPA was also radiolabeled withyttrium-[90] and its biological distribution evaluated in BALB/c miceover a 72-hour time period. Following purification by HPLC sizeexclusion chromatography, four groups of five mice each were injectedintravenously with approximately 1 μCi of clinically-formulatedconjugate (specific activity:12.2 mCi/mg); groups were subsequentlysacrificed at 1, 24, 48 and 72 hours and their organs and tissuesanalyzed as described above. Radioactivity associated with each tissuespecimen was determined by measuring bremstrahlung energy with a gammascintillation counter. Activity values were subsequently expressed aspercent injected dose per gram tissue or percent injected dose perorgan. While organs and other tissues were rinsed repeatedly to removesuperficial blood, the organs were not perfused. Thus, organ activityvalues were not discounted for the activity contribution represented byinternally associated blood.

[0094] ii. Tumor Localization of I2B8

[0095] The localization of radiolabeled 2B8-MX-DTPA was determined inathymic mice bearing Ramos B cell tumors. Six-to-eight week old athymicmice were injected subcutaneously (left-rear flank) with 0.1 mL ofRPMI-1640 containing 1.2×10⁷ Ramos tumor cells which had been previouslyadapted for growth in athymic mice. Tumors arose within two weeks andranged in weight from 0.07 to 1.1 grams. Mice were injectedintravenously with 100 μL of indium-[111]-labeled 2B8-MX-DTPA (16.7 μCi)and groups of three mice were sacrificed by cervical dislocation at 0,24, 48, and 72 hours. After sacrifice the tail, heart, lungs, liver,kidney, spleen, muscle, femur, and tumor were removed, washed, weighed;a sample of blood was also removed for analysis. Radioactivityassociated with each specimen was determined by gamma counting and thepercent injected dose per gram tissue determined.

[0096] iii. Biodistribution and Tumor Localization Studies withRadiolabeled 2B8-MX-DTPA

[0097] Following the preliminary biodistribution experiment describedabove (Example I.B.viii.a.), conjugated 2B8 was radiolabeled withindium-[111] to a specific activity of 2.3 mCi/mg and roughly 1.1 μCiwas injected into each of twenty BALB/c mice to determinebiodistribution of the radiolabeled material. Subsequentially, groups offive mice each were sacrificed at 1, 24, 48 and 72 hours and theirorgans and a portion of the skin, muscle and bone were removed andprocessed for analysis. In addition, the urine and feces were collectedand analyzed for the 24-72 hour time-points. The level of radioactivityin the blood dropped from 40.3% of the injected dose per gram at 1 hourto 18.9% at 72 hours (data not shown). Values for the heart, kidney,muscle and spleen remained in the range of 0.7-9.8% throughout theexperiment. Levels of radioactivity found in the lungs decreased from14.2% at 1 hour to 7.6% at 72 hours; similarly the respective liverinjected-dose per gram values were 10.3% and 9.9%. These data were usedin determining radiation absorbed dose estimates I2B8 described below.

[0098] The biodistribution of yttrium-[90]-labeled conjugate, having aspecific activity of 12.2 mCi/mg antibody, was evaluated in BALB/c mice.Radioincorporations of >90% were obtained and the radiolabeled antibodywas purified by HPLC. Tissue deposition of radioactivity was evaluatedin the major organs, and the skin, muscle, bone, and urine and fecesover 72 hours and expressed as percent injected dose/g tissue. Results(not shown) evidenced that while the levels of radioactivity associatedwith the blood dropped from approximately 39.2% injected dose per gramat 1 hour to roughly 15.4% after 72 hours the levels of radioactivityassociated with tail, heart, kidney, muscle and spleen remained fairlyconstant at 10.2% or less throughout the course of the experiment.Importantly, the radioactivity associated with the bone ranged from 4.4%of the injected dose per gram bone at 1 hour to 3.2% at 72 hours. Takentogether, these results suggest that little free yttrium was associatedwith the conjugate and that little free radiometal was released duringthe course of the study. These data were used in determining radiationabsorbed dose estimates for Y2B8 described below.

[0099] For tumor localization studies, 2B8-MX-DTPA was prepared andradiolabeled with ¹¹¹Indium to a specific activity of 2.7 mCi/mg. Onehundred microliters of labeled conjugate (approximately 24 μCi) weresubsequently injected into each of 12 athymic mice bearing Ramos B celltumors. Tumors ranged in weight from 0.1 to 1.0 grams. At time points of0, 24, 48, and 72 hours following injection, 50 μL of blood was removedby retro-orbital puncture, the mice sacrificed by cervical dislocation,and the tail, heart, lungs, liver, kidney, spleen, muscle, femur, andtumor removed. After processing and weighing the tissues, theradioactivity associated with each tissue specimen was determined usinga gamma counter and the values expressed as percent injected dose pergram.

[0100] The results (not shown) evidenced that the tumor concentrationsof the ¹¹¹In-2B8-MX-DTPA increased steadily throughout the course of theexperiment. Thirteen percent of the injected dose was accumulated in thetumor after 72 hours. The blood levels, by contrast, dropped during theexperiment from over 30% at time zero to 13% at 72 hours. All othertissues (except muscle) contained between 1.3 and 6.0% of the injecteddose per gram tissue by the end of the experiment; muscle tissuecontained approximately 13% of the injected dose per gram.

[0101] D. Human Studies

[0102] i. 2B8 and 2B8-MX-DTPA: Immunohistology Studies with HumanTissues

[0103] The tissue reactivity of murine monoclonal antibody 2B8 wasevaluated using a panel of 32 different human tissues fixed withacetone. Antibody 2B8 reacts with the anti-CD20 antigen which had a veryrestricted pattern of tissue distribution, being observed only in asubset of cells in lymphoid tissues including those of hematopoieticorigin.

[0104] In the lymph node, immunoreactivity was observed in a populationof mature cortical B-lymphocytes as well as proliferating cells in thegerminal centers. Positive reactivity was also observed in theperipheral blood, B-cell areas of the tonsils, white pulp of the spleen,and with 40-70% of the medullary lymphocytes found in the thymus.Positive reactivity was also seen in the follicles of the lamina propria(Peyer's Patches) of the large intestines. Finally, aggregates orscattered lymphoid cells in the stroma of various organs, including thebladder, breast, cervix, esophagus, lung, parotid, prostate, smallintestine, and stomach, were also positive with antibody 2B8 (data notshown).

[0105] All simple epithelial cells, as well as the stratified epitheliaand epithelia of different organs, were found to be unreactive.Similarly, no reactivity was seen with neuroectodermal cells, includingthose in the brain, spinal cord and peripheral nerves. Mesenchymalelements, such as skeletal and smooth muscle cells, fibroblasts,endothelial cells, and polymorphonuclear inflammatory cells were alsofound to be negative (data not shown).

[0106] The tissue reactivity of the 2B8-MX-DTPA conjugate was evaluatedusing a panel of sixteen human tissues which had been fixed withacetone. As previously demonstrated with the native antibody (data notshown), the 2B8-MX-DTPA conjugate recognized the CD20 antigen whichexhibited a highly restricted pattern of distribution, being found onlyon a subset of cells of lymphoid origin. In the lymph node,immunoreactivity was observed in the B cell population. Strongreactivity was seen in the white pulp of the spleen and in the medullarylymphocytes of the thymus. Immunoreactivity was also observed inscattered lymphocytes in the bladder, heart, large intestines, liver,lung, and uterus, and was attributed to the presence of inflammatorycells present in these tissues. As with the native antibody, noreactivity was observed with neuroectodermal cells or with mesenchymalelements (data not shown).

[0107] ii. Clinical Analysis of I2B8 (Imaging) and Y2B8 (Therapy)

[0108] a. Phase I/II Clinical Trial Single Dose Therapy Study

[0109] A Phase I/II clinical analysis of I2B8 (imaging) followed bytreatment with a single therapeutic dose of Y2B8 is currently beingconducted. For the single-dose study, the following schema is beingfollowed:

[0110] 1. Peripheral Stem Cell (PSC) or Bone Marrow (BM) Harvest withPurging;

[0111] 2. I2B8 Imaging;

[0112] 3. Y2B8 Therapy (three Dose Levels); and

[0113] 4. PSC or Autologous BM Transplantation (if necessary based uponabsolute neutrophil count below 500/mm³ for three consecutive days orplatelets below 20,000/mm³ with no evidence of marrow recovery on bonemarrow examination).

[0114] The Dose Levels of Y2B8 are as follows: Dose Level Dose (mCi) 1.20 2. 30 3. 40

[0115] Three patients are to be treated at each of the dose levels fordetermination of a Maximum Tolerated Dose (“MTD”).

[0116] Imaging (Dosimetry) Studies are conducted as follows: eachpatient is involved in two in vivo biodistribution studies using I2B8.In the first study, 2 mg of I2B8 (5 mCi), is administered as anintravenous (i.v.) infusion over one hour; one week later 2B8 (ieunconjugated antibody) is administered by i.v. at a rate not to exceed250 mg/hr followed immediately by 2 mg of I2B8 (5 mCi) administered byi.v. over one hour. In both studies, immediately following the I2B8infusion, each patient is imaged and imaging is repeated at time t=14-18hr (if indicated), t=24 hr; t=72 hr; and t=96 hr (if indicated). Wholebody average retention times for the indium [111] label are determined;such determinations are also made for recognizable organs or tumorlesions (“regions of interest”).

[0117] The regions of interest are compared to the whole bodyconcentrations of the label; based upon this comparison, an estimate ofthe localization and concentration of Y2B8 can be determined usingstandard protocols. If the estimated cumulative dose of Y2B8 is greaterthan eight (8) times the estimated whole body dose, or if the estimatedcumulative dose for the liver exceeds 1500 cGy, no treatment with Y2B8should occur.

[0118] If the imaging studies are acceptible, either 0.0 or 1.0 mg/kgpatient body weight of 2B8 is administered by i.v. infusion at a ratenot to exceed 250 mg/h. This is followed by administration of Y2B8(10,20 or 400 mCi) at an i.v. infusion rate of 20 mCi/hr.

[0119] b. Phase I/II Clinical Trial: Multiple Dose Therapy Study

[0120] A Phase I/II clinical analysis of of Y2B8 is currently beingconducted. For the multiple-dose study, the following schema is beingfollowed:

[0121] 1. PSC or BM Harvest;

[0122] 2. I2B8 Imaging;

[0123] 3. Y2B8 Therapy (three Dose Levels) for four doses or a totalcumulative dose of 80 mCi; and

[0124] 4. PSC or Autologous BM Transplantation (based upon decision ofmedical practitioner).

[0125] The Dose Levels of Y2B8 are as follows: Dose Level Dose (mCi) 1.10 2. 15 3. 20

[0126] Three patients are to be treated at each of the dose levels fordetermination of an MTD.

[0127] Imaging (Dosimetry) Studies are conducted as follows: A preferredimaging dose for the unlabeled antibody (ie 2B8) will be determined withthe first two patients. The first two patients will receive 100 mg ofunlabeled 2B8 in 250 cc of normal saline over 4 hrs followed by 0.5 mCiof I2B8—blood will be sampled for biodistribution data at times t=0,t=10 min., t=120 min., t=24 hr, and t=48 hr. Patients will be scannedwith multiple regional gamma camera images at times t=2 hr, t=24 hr andt=48 hr. After scanning at t=48 hr, the patients will receive 250 mg of2B8 as described, followed by 4.5 mCi of I2B8—blood and scanning willthen follow as described. If 100 mg of 2B8 produces superior imaging,then the next two patients will receive 50 mg of 2B8 as described,followed by 0.5 mCi of I2B8 followed 48 hrs later by 100 mg 2B8 and thenwith 4.5 mCi of I2B8. If 250 mg of 2B8 produces superior imaging, thenthe next two patients will receive 250 mg of 2B8 as described, followedby 0.5 mCi of I2B8 followed 48 hrs later with 500 mg 2B8 and then with4.5 mCi of I2B8. Subsequent patients will be treated with the lowestamount of 2B8 that provides optimal imaging. Optimal imaging will bedefined by: (1) best effective imaging with the slowest disappearance ofantibody; (2) best distribution minimizing compartmentalization in asingle organ; and (3) best subjective resolution of the lesion(tumor/background comparison).

[0128] For the first four patients, the first therapeutic dose of Y2B8will begin 14 days after the last dose of I2B8; for subsequent patients,the first therapeutic dose of Y2B8 will begin between two to seven daysafter the I2B8.

[0129] Prior to treatment with Y2B8, for the patients other than thefirst four, 2B8 will be administered as described, followed by i.v.infusion of Y2B8 over 5-10 min. Blood will be sampled forbiodistribution at times t=0, t=10 min., t=120 min., t=24 hr and t=48hr. Patients will receive repetitive doses of Y2B8 (the same doseadministered as with the first dose) approximately every six to eightweeks for a maximum of four doses, or total cumulative dose of 80 mCi.It is most preferred that patients not receive a subsequent dose of Y2B8until the patients' WBC is greater than/equal to 3,000 and AGC isgreater than/equal to 100,000.

[0130] Following completion of the three-dose level study, an MTD willbe defined. Additional patients will then be enrolled in the study andthese will receive the MTD.

II. CHIMERIC ANTI-CD20 ANTIBODY PRODUCTION (“C2B8”)

[0131] A. Construction of Chimeric Anti-CD20 Immunoglobulin DNAExpression Vector

[0132] RNA was isolated from the 2B8 mouse hybridoma cell (as describedin Chomczynki, P. et al., “Single step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction.” Anal. Biochem.162:156-159 (1987)). and cDNA was prepared therefrom. The mouseimmunoglobulin light chain variable region DNA was isolated from thecDNA by polymerase chain reaction using a set of DNA primers withhomology to mouse light chain signal sequences at the 5′ end and mouselight chain J region at the 3′ end. Primer sequences were as follows:

[0133] 1. V_(L) Sense (SEQ. ID. NO.3) 5′ ATC AC AGATCT CTC ACC ATG GATTTT CAG GTG CAG ATT ATC AGC TTC 3′

[0134] (The underlined portion is a Bgl II site; the above-lined portionis the start codon.)

[0135] 2. V_(L) Antisense (SEQ. ID. NO.4) 5′ TGC AGC ATC CGTACG TTT GATTTC CAG CTT 3′

[0136] (The underlined portion is a Bsi WI site.)

[0137] See, FIGS. 1 and 2 for the corresponding Bgl II and Bsi WI sitesin TCAE 8, and FIG. 3 for the corresponding sites in anti-CD20 in TCAE8.

[0138] These resulting DNA fragment was cloned directly into the TCAE 8vector in front of the human kappa light chain constant domain andsequenced. The determined DNA sequence for the murine variable regionlight chain is set forth in FIG. 4 (SEQ. ID. NO. 5); see also FIG. 3,nucleotides 978 through 1362. FIG. 4 further provides the amino acidsequence from this murine variable region, and the CDR and frameworkregions. The mouse light chain variable region from 2B8 is in the mousekappa VI family. See, Kabat, supra.

[0139] The mouse heavy chain variable region was similarly isolated andcloned in front of the human IgGl constant domains. Primers were asfollows: 1. V_(H) Sense (SEQ. ID. NO.6) 5′ GCG GCT CCC ACGCGT GTC CTGTCC CAG 3′

[0140] (The underlined portion is an Mlu I site.) 2. V_(H) Antisense(SEQ. ID. NO.7) 5′ GG(G/C) TGT TGT GCTAGC TG(A/C) (A/G)GA GAC (G/A)GTGA 3′

[0141] (The underlined portion is an Nhe I site.)

[0142] See, FIGS. 1 and 2 for corresponding Mlu I and Nhe I sites inTCAE 8, and FIG. 3 for corresponding sites in anti-CD20 in TCAE 8.

[0143] The sequence for this mouse heavy chain is set forth in FIG. 5(SEQ. ID. NO. 8); see also FIG. 3, nucleotide 2401 through 2820. FIG. 5also provides the amino acid sequence from this murine variable region,and the CDR and framework regions. The mouse heavy chain variable regionfrom 2B8 is in the mouse VH 2B family. See, Kabat, supra.

[0144] B. Creation of Chimeric Anti-CD20 Producing CHO and SP2/0Transfectomas

[0145] Chinese hamster ovary (“CHO”) cells DG44 were grown in SSFM IIminus hypoxanthine and thymidine media (Gibco, Grand Island, N.Y., FormNo. 91-0456PK); SP2/0 mouse myeloma cells were grown in Dulbecco'sModified Eagles Medium media (“DMEM”) (Irvine Scientific, Santa Ana,Calif., Cat. No. 9024) with 5% fetal bovine serum and 20 ml/L glutamineadded. Four million cells were electroporated with either 25 μg CHO or50 μg SP2/0 plasmid DNA that had been restricted with Not I using a BTX600 electroporation system (BTX, San Diego, Calif.) in 0.4 ml disposablecuvettes. Conditions were either 210 volts for CHO or 180 volts forSP2/0, 400 microfaradays, 13 ohms. Each electroporation was plated intosix 96 well dishes (about 7,000 cells/well). Dishes were fed with mediacontaining G418 (GENETICIN, Gibco, Cat. No.860-1811) at 400 μg/ml activecompound for CHO (media further included 50 μM hypoxanthine and 8 μMthymidine) or 800 μg/ml for SP2/0, two days following electroporationand thereafter 2 or 3 days until colonies arose. Supernatant fromcolonies was assayed for the presence of chimeric immunoglobulin via anELISA specific for human antibody. Colonies producing the highest amountof immunoglobulin were expanded and plated into 96 well platescontaining media plus methotrexate (25 nM for SP2/0 and 5 nM for CHO)and fed every two or three days. Supernatants were assayed as above andcolonies producing the highest amount of immunoglobulin were examined.Chimeric anti-CD20 antibody was purified from supernatant using proteinA affinity chromatography.

[0146] Purified chimeric anti-CD20 was analyzed by electrophoresis inpolyacrylamide gels and estimated to be greater than about 95% pure.Affinity and specificity of the chimeric antibody was determined basedupon 2B8. Chimeric anti-CD20 antibody tested in direct and competitivebinding assays, when compared to murine anti-CD20 monoclonal antibody2B8, evidenced comparable affinity and specificity on a number of CD20positive B cells lines (data not presented). The apparent affinityconstant (“Kap”) of the chimeric antibody was determined by directbinding of I¹²⁵ radiolabeled chimeric anti-CD20 and compared toradiolabeled 2B8 by Scatchard plot; estimated Kap for CHO producedchimeric anti-CD20 was 5.2×10⁻⁹ M and for SP2/0 produced antibody,7.4×10⁻⁹M. The estimated Kap for 2B8 was 3.5×10⁻⁹ M. Direct competitionby radioimmunoassay was utilized to confirm both the specificity andretention of immunoreactivity of the chimeric antibody by comparing itsability to effectively compete with 2B8. Substantially equivalentamounts of chimeric anti-CD20 and 2B8 antibodies were required toproduce 50% inhibition of binding to CD20 antigens on B cells (data notpresented), ie there was a minimal loss of inhibiting activity of theanti-CD20 antibodies, presumably due to chimerization.

[0147] The results of Example II.B indicate, inter alia, that chimericanti-CD20 antibodies were generated from CHO and SP2/0 transfectomasusing the TCAE 8 vectors, and these chimeric antibodies hadsubstantially the same specificity and binding capability as murineanti-CD20 monoclonal antibody 2B8.

[0148] C. Determination of Immunological Activity of Chimeric Anti-CD20Antibodies

[0149] i. Human C1q Analysis

[0150] Chimeric anti-CD20 antibodies produced by both CHO and SP2/0 celllines were evaluated for human C1q binding in a flow cytometry assayusing fluorescein labeled C1q (C1q was obtained from Quidel, Mira Mesa,Calif., Prod. No. A400 and FITC label from Sigma, St. Louis Mo., Prod.No. F-7250; FITC. Labeling of C1q was accomplished in accordance withthe protocol described in Selected Methods In Cellular Immunology,Michell & Shiigi, Ed. (W. H. Freeman & Co., San Francisco, Calif., 1980,p. 292). Analytical results were derived using a Becton DickinsonFACScan™ flow cytometer (fluorescein measured over a range of 515-545nm). Equivalent amounts of chimeric anti-CD20 antibody, human IgG1,Kmyeloma protein (Binding Site, San Diego, Calif., Prod. No. BP078), and2B8 were incubated with an equivalent number of CD20-positive SB cells,followed by a wash step with FACS buffer (0.2% BSA in PBS, pH 7.4, 0.02%sodium azide) to remove unattached antibody, followed by incubation withFITC labeled C1q. Following a 30-60 min. incubation, cells were againwashed. The three conditions, including FITC-labeled C1q as a control,were analyzed on the FACScan™ following manufacturing instructions.Results are presented in FIG. 6.

[0151] As the results of FIG. 6 evidence, a significant increase influorescence was observed only for the chimeric anti-CD20 antibodycondition; ie only SB cells with adherent chimeric anti-CD20 antibodywere C1q positive, while the other conditions produced the same patternas the control.

[0152] ii. Complement Dependent Cell Lyses

[0153] Chimeric anti-CD20 antibodies were analyzed for their ability tolyse lymphoma cell lines in the presence of human serum (complementsource). CD20 positive SB cells were labeled with ⁵¹Cr by admixing 100 μCi of ⁵¹Cr with 1×10⁶ SB cells for 1 hr at 37° C.; labeled SB cells werethen incubated in the presence of equivalent amounts of human complementand equivalent amounts (0-50 μg/ml) of either chimeric anti-CD20antibodies or 2B8 for 4 hrsat 37° C. (see, Brunner. K. T. et al.,“Quantitative assay of the lytic action of immune lymphoid cells on⁵¹Cr-labeled allogeneic target cells in vitro.” Immunology 14:181-189(1968). Results are presented in FIG. 7.

[0154] The results of FIG. 7 indicate, inter alia, that chimericanti-CD20 antibodies produced significant lysis (49%) under theseconditions.

[0155] iii. Antibody Dependent Cellular Cytotoxicity Effector Assay

[0156] For this study, CD20 positive cells (SB) and CD20 negative cells(T cell leukemia line HSB; see, Adams, Richard, “Formal Discussion,”Can. Res. 27:2479-2482 (1967); ATCC deposit no. ATCC CCL 120.1) wereutilized; both were labeled with ⁵¹Cr. Analysis was conducted followingthe protocol described in Brunner, K. T. et al., “Quantitative assay ofthe lytic action of immune lymphoid cells on ⁵¹Cr-labeled allogeneictarget cells in vitro; inhibition by isoantibody and drugs.” Immunology14:181-189 (1968); a substantial chimeric anti-CD20 antibody dependentcell mediated lysis of CD20 positive SB target cells (⁵¹Cr-labeled) atthe end of a 4 hr, 37° C. incubation, was observed and this effect wasobserved for both CHO and SP2/0 produced antibody (effector cells werehuman peripheral lymphocytes; ratio of effector cells:target was 100:1).Efficient lysis of target cells was obtained at 3.9 μg/ml. In contrast,under the same conditions, the murine anti-CD20 monoclonal antibody 2B8had a statistically insignificant effect, and CD20 negative HSB cellswere not lysed. Results are presented in FIG. 8.

[0157] The results of Example II indicate, inter alia, that the chimericanti-CD20 antibodies of Example I were immunologically active.

III. DEPLETION OF B CELLS IN VIVO USING CHIMERIC ANTI-CD20

[0158] A. Non-Human Primate Study

[0159] Three separate non-human primate studies were conducted. Forconvenience, these are referred to herein as “Chimeric Anti-CD20: CHO &SP2/0;” “Chimeric Anti-CD20: CHO;” and “High Dosage Chimeric Anti-CD20.”Conditions were as follows:

[0160] Chimeric Anti-CD20: CHO & SP2/0

[0161] Six cynomolgus monkeys ranging in weight from 4.5 to 7 kilograms(White Sands Research Center, Alamogordo, N.Mex.) were divided intothree groups of two monkeys each. Both animals of each group receivedthe same dose of immunologically active chimeric anti-CD20 antibody. Oneanimal in each group received purified antibody produced by the CHOtransfectoma; the other received antibody produced by the SP2/0transfectoma. The three groups received antibody dosages correspondingto 0.1 mg/kg, 0.4 mg/kg, and 1.6 mg/kg each day for four (4) consecutivedays. The chimeric immunologically active anti-CD20 antibody, which wasadmixed with sterile saline, was administered by intravenous infusion;blood samples were drawn prior to each infusion. Additional bloodsamples were drawn beginning 24 hrs after the last injection (T=O) andthereafter on days 1, 3, 7, 14 and 28; blood samples were also takenthereafter at biweekly intervals until completion of the study at day90.

[0162] Approximately 5 ml of whole blood from each animal wascentrifuged at 2000 RPM for 5 min. Plasma was removed for assay ofsoluble chimeric anti-CD20 antibody levels. The pellet (containingperipheral blood leukocytes and red blood cells) was resuspended infetal calf serum for fluorescent-labeled antibody analysis (see,“Fluorescent Antibody Labeling of Lymphoid Cell Population,” infra.).

[0163] Chimeric Anti-CD20: CHO

[0164] Six cynomolgus monkeys ranging in weight from 4 to 6 kilograms(White Sands) were divided into three groups of two monkeys each. Allanimals were injected with immunologically active chimeric anti-CD20antibodies produced from the CHO transfectoma (in sterile saline). Thethree groups were separated as follows: subgroup 1 received dailyintravenous injections of 0.01 mg/kg of the antibody over a four (4) dayperiod; subgroup 2 received daily intravenous injections of 0.4 mg/kg ofthe antibody over a four (4) day period; subgroup 3 received a singleintravenous injection of 6.4 mg/kg of the antibody. For all threesubgroups, a blood sample was obtained prior to initiation of treatment;additionally, blood samples were also drawn at T=0, 1, 3, 7, 14 and 28days following the last injection, as described above, and these sampleswere processed for fluorescent labeled antibody analysis (see,“Fluorescent Antibody Labeling,” infra.). In addition to peripheralblood B cell quantitation, lymph node biopsies were taken at days 7, 14and 28 following the last injection, and a single cell preparationstained for quantitation of lymphocyte populations by flow cytometry.

[0165] High Dosage Chimeric Anti-CD20

[0166] Two cynomolgus monkeys (White Sands) were infused with 16.8 mg/kgof the immunologically active chimeric anti-CD20 antibodies from the CHOtransfectomas (in sterile saline) weekly over a period of fourconsecutive weeks. At the conclusion of the treatment, both animals wereanesthetized for removal of bone marrow; lymph node biopsies were alsotaken. Both sets of tissue were stained for the presence of Blymphocytes using Leu 16 by flow cytometry following the protocoldescribed in Ling, N. R. et al., “B-cell and plasma cell antigens.”Leucocyte Typing III White Cell Differentiations Antigens, A. J.McMichael, Ed. (Oxford University Press, Oxford UK, 1987), p. 302.

[0167] Fluorescent Antibody Labeling of Lymphoid Cell Population

[0168] After removal of plasma, leukocytes were washed twice with HanksBalanced Salt Solution (“HBSS”) and resuspended in a plasma equivalentvolume of fetal bovine serum (heat inactivated at 56° C. for 30 min.). A0.1 ml volume of the cell preparation was distributed to each of six(6), 15 ml conical centrifuge tubes Fluorescein labeled monoclonalantibodies with specificity for the human lymphocyte surface markers CD2(AMAC, Westbrook, Me.), CD20 (Becton Dickinson) and human IgM (BindingSite, San Diego, Calif.) were added to 3 of the tubes for identifying Tand B lymphocyte populations. All reagents had previously testedpositive to the corresponding monkey lymphocyte antigens. Chimericanti-CD20 antibody bound to monkey B cell surface CD20 was measured inthe fourth tube using polyclonal goat anti-human IgG coupled withphycoerythrin (AMAC). This reagent was pre-adsorbed on a monkeyIg-sepharose column to prevent cross-reactivity to monkey Ig, thusallowing specific detection and quantitation of chimeric anti-CD20antibody bound to cells. A fifth tube included both anti-IgM andanti-human IgG reagents for double stained B cell population. A sixthsample was included with no reagents for determination ofautofluorescence. Cells were incubated with fluorescent antibodies for30 min., washed and fixed with 0.5 ml of fixation buffer (0.15 M NaCl,1% paraformaldehyde, pH7.4) and analyzed on a Becton Dickinson FACScan™instrument. Lymphocyte populations were initially identified by forwardversus right angle light scatter in a dot-plot bitmap with unlabeledleucocytes. The total lymphocyte population was then isolated by gatingout all other events. Subsequent fluorescence measurements reflectedonly gated lymphocyte specific events.

[0169] Depletion of Peripheral Blood B Lymphocytes

[0170] No observable difference could be ascertained between theefficacy of CHO and SP2/0 produced antibodies in depleting B cells invivo, although a slight increase in B cell recovery beginning after day7 for monkeys injected with chimeric anti-CD20 antibodies derived fromCHO transfectomas at dosage levels 1.6 mg/kg and 6.4 mg/kg was observedand for the monkey injected with SP2/0 producing antibody at the 0.4mg/kg dose level. FIGS. 9A, B and C provide the results derived from thechimeric anti-CD20:CHO & SP2/0 study, with FIG. 9A directed to the 0.4mg/kg dose level; FIG. 9B directed to the 1.6 mg/kg dose level; and FIG.9C directed to the 6.4 mg/kg dose level.

[0171] As is evident from FIG. 9, there was a dramatic decrease (>95%)in peripheral B cell levels after the therapeutic treatment across alltested dose ranges, and these levels were maintained up to seven (7)days post infusion; after this period, B cell recovery began, and, thetime of recovery initiation was independent of dosage levels.

[0172] In the Chimeric Anti-CD20:CHO study, a 10-fold lower antibodydosage concentration (0.01 mg/kg) over a period of four daily injections(0.04 mg/kg total) was utilized. FIG. 10 provides the results of thisstudy. This dosage depleted the peripheral blood B cell population toapproximately 50% of normal levels estimated with either theanti-surface IgM or the Leu 16 antibody. The results also indicate thatsaturation of the CD20 antigen on the B lymphocyte population was notachieved with immunologically active chimeric anti-CD20 antibody at thisdose concentration over this period of time for non-human primates; Blymphocytes coated with the antibody were detected in the blood samplesduring the initial three days following therapeutic treatment. However,by day 7, antibody coated cells were undetectable.

[0173] Table I summarizes the results of single and multiple doses ofimmunologically active chimeric anti-CD20 antibody on the peripheralblood populations; single dose condition was 6.4 mg/kg; multiple dosecondition was 0.4 mg/kg over four (4) consecutive days (these resultswere derived from the monkeys described above). TABLE I PERIPHERAL BLOODPOPULATION FROM C2B8 PRIMATE STUDY Monkey Dose Day CD2 Anti-Hu IgG A 0.4mg/kg Prebleed 81.5 — (4 doses)  0 86.5 0.2  7 85.5 0.0 21 93.3 — 2885.5 — B 0.4 mg/kg Prebleed 81.7 — (4 doses)  0 94.6 0.1  7 92.2 0.1 2184.9 — 28 84.1 — C 6.4 mg/kg Prebleed 77.7 0.0 (1 dose)  7 85.7 0.1 2186.7 — 28 76.7 — D 6.4 mg/kg Prebleed 85.7 0.1 (1 dose)  7 94.7 0.1 2185.2 — 28 85.9 — Anti-Hu IgG + Monkey Anti-Hu IgM* Leu-16 % B CellDepletion A — 9.4  0 0.3 0.0 97 0.1 1.2 99 — 2.1 78 — 4.1 66 B — 14.8  00.2 0.1 99 0.1 0.1 99 — 6.9 53 — 8.7 41 C 0.2 17.0  0 0.1 0.0 99 — 14.715 — 8.1 62 D 0.1 14.4  0 0.2 0.0 99 — 9.2 46 — 6.7 53

[0174] The data summarized in Table I indicates that depletion of Bcells in peripheral blood under conditions of antibody excess occurredrapidly and effectively, regardless of single or multiple dosage levels.Additionally, depletion was observed for at least seven (7) daysfollowing the last injection, with partial B cell recovery observed byday 21.

[0175] Table II summarizes the effect of immunologically active,chimeric anti-CD20 antibodies on cell populations of lymph nodes usingthe treatment regimen of Table I (4 daily doses of 0.4 mg/kg; 1 dose of6.4 mg/kg); comparative values for normal lymph nodes (control monkey,axillary and inguinal) and normal bone marrow (two monkeys) are alsoprovided. TABLE II CELL POPULATIONS OF LYMPH NODES Monkey Dose Day CD2Anti-Hu IgM A 0.4 mg/kg  7 66.9 — (4 doses) 14 76.9 19.6 28 61.6 19.7 B0.4 mg/kg  7 59.4 — (4 doses) 14 83.2  9.9 28 84.1 15.7 C 6.4 mg/kg  775.5 — (1 dose) 14 74.1 17.9 28 66.9 23.1 D 6.4 mg/kg  7 83.8 — (1 dose)14 74.1 17.9 28 84.1 12.8 Anti-Hu IgG + Monkey Anti-Hu IgM* Leu-16 % BLymphocyte Depletion A  7.4 40.1  1  0.8 22.6 44 — 26.0 36 B 29.9 52.2 0  0.7 14.5 64 — 14.6 64 C 22.3 35.2 13  1.1 23.9 41 — 21.4 47 D 12.519.7 51  0.2  8.7 78 — 12.9 68 Anti-Hu IgG + Anti-Hu % B Lymphocyte CD2Anti-Hu IgM IgM Leu-16 Depletion Normal Lymph Nodes Control 1 55.4 25.0— 41.4 NA Axillary 52.1 31.2 — 39.5 NA Inguinal Normal Bone MarrowControl 2 65.3 19.0 — 11.4 NA Control 3 29.8 28.0 — 16.6 NA

[0176] The results of Table II evidence effective depletion of Blymphocytes for both treatment regimens. Table II further indicates thatfor the non-human primates, complete saturation of the B cells in thelymphatic tissue with immunologically active, chimeric anti-CD20antibody was not achieved; additionally, antibody coated cells wereobserved seven (7) days after treatment, followed by a marked depletionof lymph node B cells, observed on day 14.

[0177] Based upon this data, the single High Dosage Chimeric Anti-CD20study referenced above was conducted, principally with an eye towardpharmacology/toxicology determination. Ie this study was conducted toevaluate any toxicity associated with the administration of the chimericantibody, as well as the efficacy of B cell depletion from peripheralblood lymph nodes and bone marrow. Additionally, because the data ofTable II indicates that for that study, the majority of lymph node Bcells were depleted between 7 and 14 days following treatment, a weeklydosing regimen might evidence more efficacious results. Table IIIsummarizes the results of the High Dosage Chimeric Anti-CD20 study.TABLE III CELL POPULATIONS OF LYMPH NODES AND BONE MARROW LymphocytePopulations (%) Monkey CD2 CD20^(a) mIgM + anti-C2B8^(b) C2B8^(c)Day^(d) Inguinal Lymph Node E 90.0 5.3 4.8 6.5 22 F 91.0 6.3 5.6 6.3 22G 89.9 5.0 3.7 5.8 36 H 85.4 12.3  1.7 1.8 36 Bone Marrow E 46.7 4.3 2.62.8 22 F 41.8 3.0 2.1 2.2 22 G 35.3 0.8 1.4 1.4 36 H 25.6 4.4 4.3 4.4 36

[0178] Both animals evaluated at 22 days post treatment cessationcontained less than 5% B cells, as compared to 40% in control lymphnodes (see, Table II, supra). Similarly, in the bone marrow of animalstreated with chimeric anti-CD20 antibody, the levels of CD20 positivecells were less than 3% as compared to 11-15% in the normal animals(see, Table II, supra). In the animals evaluated at 36 days posttreatment cessation, one of the animals (H) had approximately 12% Bcells in the lymph node and 4.4% B cells in bone marrow, while the other(G) had approximately 5% B cells in the lymph node and 0.8% in the bonemarrow—the data is indicative of significant B cell depletion.

[0179] The results of Example III,A indicate, inter alia that low dosesof immunologically active, chimeric anti-CD20 leads to long-termperipheral blood B cell depletion in primates. The data also indicatesthat significant depletion of B cell populations was achieved inperipheral lymph nodes and bone marrow when repetitive high doses of theantibody were administered. Continued follow-up on the test animals hasindicated that even with such severe depletion of peripheral Blymphocytes during the first week of treatment, no adverse healtheffects have been observed. Furthermore, as recovery of B cellpopulation was observed, a conclusion to be drawn is that thepluripotent stem cells of these primates were not adversely affected bythe treatment.

[0180] B. Clinical Analysis of C2B8

[0181] i. Phase I/II Clinical Trial of C2B8: Single Dose Therapy Study

[0182] Fifteen patients having histologically documented relapsed B celllymphoma have been treated with C2B8 in a Phase I/II Clinical Trial.Each patient received a single dose of C2B8 in a dose-escalating study;there were three patients per dose: 10 mg/m²; 50 mg/m²; 100 mg/m²; 250mg/m² and 500 mg/m². Treatment was by i.v. infusion through an 0.22micron in-line filter with C2B8 being diluted in a final volume of 250cc or a maximal concentration of 1 mg/ml of normal saline. Initial ratewas 50 cc/hr for the first hour; if no toxicity was seen, dose rate wasable to be escalated to a maximum of 200 cc/hr.

[0183] Toxicity (as indicated by the clinician) ranged from “none”, to“fever” to “moderate” (two patients) to “severe” (one patient); allpatients completed the therapy treatment. Peripheral Blood Lymphocyteswere analyzed to determine, inter alia, the impact of C2B8 on T-cellsand B-cells. Consistently for all patients, Peripheral Blood BLymphocytes were depleted after infusion with C2B8 and such depletionwas maintained for in excess of two weeks.

[0184] One patient (receiving 100 mg/² of C2B8) evidenced a PartialResponse to the C2B8 treatment (reduction of greater than 50% in the sumof the products of the perpendicular diameters of all measurableindicator lesions lasting greater than four weeks, during which no newlesions may appear and no existing lesions may enlarge); at least oneother patient (receiving 500 mg/m²) evidenced a Minor Response to theC2B8 treatment (reduction of less than 50% but at least 25% in the sumof the products of the two longest perpendicular diameters of allmeasurable indicator lesions). For presentational efficiency, results ofthe PBLs are set forth in FIG. 14; data for the patient evidencing a PRis set forth in FIG. 14A; for the patient evidencing an MR, data is setforth in FIG. 14B. In FIG. 14, the following are applicable:

=Lymphocytes;

=CD3+cells (T cells);

=CD20⁺cells;

=CD19⁺cells;

=Kappa;

=lambda; and

=C2B8. As evidenced, the B cell markers CD20 and CD19, Kappa and Lambda,were depleted for a period in excess of two weeks; while there was aslight, initial reduction in T-cell counts, these returned to anapproximate base-line level in a relatively rapid time-frame.

[0185] ii. Phase I/II Clinical Trial of C2B8: Multiple Dose TherapyStudy

[0186] Patients having histologically confirmed B cell lymphoma withmeasurable progressive disease are eligible for this study which isseparated into two parts: in Phase I, consisting of a dose escalation tocharacterize dose limiting toxicities and determination of biologicallyactive tolerated dose level, groups of three patients will receiveweekly i.v. infusions of C2B8 for a total of four (4) separateinfusions. Cumulative dose at each of the three levels will be asfollows: 500 mg/m² (125 mg/m²/infusion); 1000 mg/m² (250mg/m²/infusion); 1500 mg/m² (375 mg/m²/infusion. A biologically activetolerated dose is defined, and will be determined, as the lowest dosewith both tolerable toxicity and adequate activity); in Phase II,additional patients will receive the biologically active tolerated dosewith an emphasis on determining the activity of the four doses of C2B8.

IV. COMBINATION THERAPY: C2B8 AND Y2B8

[0187] A combination therapeutic approach using C2B8 and Y2B8 wasinvestigated in a mouse xenographic model (nu/nu mice, female,approximately 10 weeks old) utilizing a B cell lymphoblastic tumor(Ramos tumor cells). For comparative purposes, additional mice were alsotreated with C2B8 and Y2B8.

[0188] Ramos tumor cells (ATCC, CRL 1596) were maintained in cultureusing RPMI-1640 supplemented with 10% fetal calf serum and glutamine at37° C. and 5% CO₂. Tumors were initiated in nine female nude miceapproximately 7-10 weeks old by subcutaneous injection of 1.7×10⁶ Ramoscells in a volume of 0.10 ml (HBSS) using a 1 cc syringe fitted with 25g needle. All animals were manipulated in a laminar flow hood and allcages, bedding, food and water were autoclaved. Tumor cells werepassaged by excising tumors and passing these through a 40 mesh screen;cells were washed twice with 1×HBSS (50 ml) by centrifugation (1300RPM),resuspended in IX HBSS to 10×10⁶ cells/ml, and frozen at −70° C. untilused.

[0189] For the experimental conditions, cells from several frozen lotswere thawed, pelleted by centrifugation (1300RPM) and washed twice with1×HBSS. Cells were then resuspended to approximately 2.0×10⁶ cells/ml.Approximately 9 to 12 mice were injected with 0.10 ml of the cellsuspension (s.c.) using a 1 cc syringe fitted with a 25 g needle;injections were made on the animal's left side, approximatelymid-region. Tumors developed in approximately two weeks. Tumors wereexcised and processed as described above. Study mice were injected asdescribed above with 1.67×10⁶ cells in 0.10 ml HBSS.

[0190] Based on preliminary dosing experiments, it was determined that200 mg of C2B8 and 100 μCi of Y2B8 would be utilized for the study.Ninety female nu/nu mice (approximately 10 weeks old) were injected withthe tumor cells. Approximately ten days later, 24 mice were assigned tofour study groups (six mice/group) while attempting to maintain acomparable tumor size distribution in each group (average tumor size,expressed as a product of length×width of the tumor, was approximately80 mm²). The following groups were treated as indicated via tail-vaininjections using a 100 μl Hamilton syringe fitted with a 25 g needle: A.Normal Saline B. Y2B8 (100 μCi) C. C2B8 (200 μg); and D. Y2B8 (100μCi) + C2B8 (200 μg)

[0191] Groups tested with C2B8 were given a second C2B8 injection (200μg/mouse) seven days after the initial injection. Tumor measurementswere made every two or three days using a caliper.

[0192] Preparation of treatment materials were in accordance with thefollowing protocols:

[0193] A. Preparation of Y2B8

[0194] Yttrium-[90] chloride (6 mCi) was transformed to a polypropylenetube and adjusted to pH 4.1-4.4 using metal free 2M sodium acetate.2B8-MX-DTPA (0.3 mg in normal saline; see above for preparation of2B8-MX-DTPA) was added and gently mixed by vortexing. After 15 min.incubation, the reaction was quenched by adding 0.05×volume 20 mM EDTAand 0.05×volume 2M sodium acetate. Radioactivity concentration wasdetermined by diluting 5.0 μl of the reaction mixture in 2.5 ml 1×PBScontaining 75 mg/ml HSA and 1 mM DTPA, (“formulation buffer”); countingwas accomplished by adding 10.0 μl to 20 ml of Ecolume™ scintillationcocktail. The remainder of the reactive mixture was added to 3.0 mlformulation buffer, sterile filtered and stored at 2-8° C. until used.Specific activity (14 mCi/mg at time of injection) was calculated usingthe radioactivity concentration and the calculated protein concentrationbased upon the amount of antibody added to the reaction mixture.Protein-associated radioactivity was determined using instant thin-layerchromatography. Radioincorporation was 95%. Y2B8 was diluted informulation buffer immediately before use and sterile-filtered (finalradioactivity concentration was 1.0 mCi/ml).

[0195] B. Preparation of C2B8

[0196] C2B8 was prepared as described above. C2B8 was provided as asterile reagent in normal saline at 5.0 mg/ml. Prior to injection, theC2B8 was diluted in normal saline to 2.0 mg/ml and sterile filtered.

[0197] C. Results

[0198] Following treatment, tumor size was expressed as a product oflength and width, and measurements were taken on the days indicated inFIG. 11 (Y2B8 vs. Saline); FIG. 12 (C2B8 vs. Saline); and FIG. 13(Y2B8+C2B8 vs. Saline). Standard error was also determined.

[0199] As indicated in FIG. 13, the combination of Y2B8 and C2B8exhibited tumoricidal effects comparable to the effects evidenced byeither Y2B8 or C2B8.

V. ALTERNATIVE THERAPY STRATEGIES

[0200] Alternative therapeutic strategies recognized in view of theforegoing examples are evident. One such strategy employs the use of atherapeutic dose of C2B8 followed within about one week with acombination of either 2B8 and radioabeled 2B8 (eg Y2B8); or 2B8, C2B8and, eg Y2B8; or C2B8 and, eg Y2B8. An additional strategy isutilization of radiolabeled C2B8—such a strategy allows for utilizationof the benefits of the immunologically active portion of C2B8 plus thosebenefits associated with a radiolabel. Preferred radiolabels includeyttrium-90 given the larger circulating half-life of C2B8 versus themurine antibody 2B8. Because of the ability of C2B8 to deplete B-cells,and the benefits to be derived from the use of a radiolabel, a preferredalternative strategy is to treat the patient with C2B8 (either with asingle dose or multiple doses) such that most, if not all, peripheral Bcells have been depleted. This would then be followed with the use ofradiolabeled 2B8; because of the depletion of peripheral B cells, theradiolabeled 2B8 stands an increased chance of targeting tumor cells.Iodine [131] labeled 2B8 is preferably utilized, given the types ofresults reported in the literature with this label (see Kaminski). Analternative preference involves the use of a radiolabeled 2B8 (or C2B8)first in an effort to increase the permeability of a tumor, followed bysingle or multiple treatments with C2B8; the intent of this strategy isto increase the chances of the C2B8 in getting both outside and insidethe tumor mass. A further strategy involved the use of chemotherapeuticagenst in combination with C2B8. These strategies include so-called“staggered” treatments, ie, treatment with chemotherapeutic agent,followed by treatment with C2B8, followed by a repetition of thisprotocol. Alternatively, initial treatment with a single or multipledoses of C2B8, thereafter followed with chemotherapeutic treatement, isviable. Preferred chemotherapeutic agents include, but are not limitedto: cyclophlsphamide; doxorubicin; vincristine; and prednisone, SeeArmitage, J. O. et al., Cancer 50:1695 (1982), incorporated herein byreference.

[0201] The foregoing alternative therapy strategies are not intended tobe limiting, but rather are presented as being representative.

VI. DEPOSIT INFORMATION

[0202] Anti-CD20 in TCAE 8 (transformed in E. coli for purposes ofdeposit) was deposited with the American Type Culture Collection (ATCC),12301 Parklawn Drive, Rockville, Md., 20852, under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure (“Budapest Treaty”).The microorganism was tested by the ATCC on Nov. 9, 1992, and determinedto be viable on that date. The ATCC has assigned this microorganism forthe following ATCC deposit number: ATCC 69119 (anti-CD20 in TCAE 8).Hybridoma 2B8 was deposited with the ATCC on Jun. 22, 1993 under theprovisions of the Budapest Treaty. The viability of the culture wasdetermined on Jun. 25, 1993 and the ATCC has assigned this hybridoma thefollowing ATCC deposit number: HB 11388.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:11 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 8541 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (viii) POSITION IN GENOME: (A)CHROMOSOME/SEGMENT: TCAE 8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCA CTACTTCTGG AATAGCTCAG 60AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA TGCATGGGGC 120GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGG GCGGAGTTAG GGGCGGGACT 180ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTC TGCCTGCTGG GGAGCCTGGG 240GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT TCTGCCTGCT 300GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACAT TCCACAGAAT TAATTCCCCT 360AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC 420GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG 480ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA 540TGGGTGGACT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA 600AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC 660ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC 720ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA 780TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG 840GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA 900CGGTGGGAGG TCTATATAAG CAGAGCTGGG TACGTGAACC GTCAGATCGC CTGGAGACGC 960CATCACAGAT CTCTCACCAT GAGGGTCCCC GCTCAGCTCC TGGGGCTCCT GCTGCTCTGG 1020CTCCCAGGTG CACGATGTGA TGGTACCAAG GTGGAAATCA AACGTACGGT GGCTGCACCA 1080TCTGTCTTCA TCTTCCCGCC ATCTGATGAG CAGTTGAAAT CTGGAACTGC CTCTGTTGTG 1140TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTAC AGTGGAAGGT GGATAACGCC 1200CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGG ACAGCAAGGA CAGCACCTAC 1260AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACG AGAAACACAA AGTCTACGCC 1320TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAA AGAGCTTCAA CAGGGGAGAG 1380TGTTGAATTC AGATCCGTTA ACGGTTACCA ACTACCTAGA CTGGATTCGT GACAACATGC 1440GGCCGTGATA TCTACGTATG ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA GCCATCTGTT 1500GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTG CCACTCCCAC TGTCCTTTCC 1560TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGT GTCATTCTAT TCTGGGGGGT 1620GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACA ATAGCAGGCA TGCTGGGGAT 1680GCGGTGGGCT CTATGGAACC AGCTGGGGCT CGACAGCTAT GCCAAGTACG CCCCCTATTG 1740ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT 1800TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT 1860GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC 1920CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 1980GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 2040TAAGCAGAGC TGGGTACGTC CTCACATTCA GTGATCAGCA CTGAACACAG ACCCGTCGAC 2100ATGGGTTGGA GCCTCATCTT GCTCTTCCTT GTCGCTGTTG CTACGCGTGT CGCTAGCACC 2160AAGGGCCCAT CGGTCTTCCC CCTGGCACCC TCCTCCAAGA GCACCTCTGG GGGCACAGCG 2220GCCCTGGGCT GCCTGGTCAA GGACTACTTC CCCGAACCGG TGACGGTGTC GTGGAACTCA 2280GGCGCCCTGA CCAGCGGCGT GCACACCTTC CCGGCTGTCC TACAGTCCTC AGGACTCTAC 2340TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC AGCAGCTTGG GCACCCAGAC CTACATCTGC 2400AACGTGAATC ACAAGCCCAG CAACACCAAG GTGGACAAGA AAGCAGAGCC CAAATCTTGT 2460GACAAAACTC ACACATGCCC ACCGTGCCCA GCACCTGAAC TCCTGGGGGG ACCGTCAGTC 2520TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC TGAGGTCACA 2580TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG GTACGTGGAC 2640GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA CAGCACGTAC 2700CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA GGACTACAAG 2760TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC CAAAGCCAAA 2820GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA GCTGACCAGG 2880AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT CGCCGTGGAG 2940TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT GCTGGACTCC 3000GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG GCAGCAGGGG 3060AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC GCAGAAGAGC 3120CTCTCCCTGT CTCCGGGTAA ATGAGGATCC GTTAACGGTT ACCAACTACC TAGACTGGAT 3180TCGTGACAAC ATGCGGCCGT GATATCTACG TATGATCAGC CTCGACTGTG CCTTCTAGTT 3240GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC 3300CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGTCATT 3360CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAA GACAATAGCA 3420GGCATGCTGG GGATGCGGTG GGCTCTATGG AACCAGCTGG GGCTCGACAG CGCTGGATCT 3480CCCGATCCCC AGCTTTGCTT CTCAATTTCT TATTTGCATA ATGAGAAAAA AAGGAAAATT 3540AATTTTAACA CCAATTCAGT AGTTGATTGA GCAAATGCGT TGCCAAAAAG GATGCTTTAG 3600AGACAGTGTT CTCTGCACAG ATAAGGACAA ACATTATTCA GAGGGAGTAC CCAGAGCTGA 3660GACTCCTAAG CCAGTGAGTG GCACAGCATT CTAGGGAGAA ATATGCTTGT CATCACCGAA 3720GCCTGATTCC GTAGAGCCAC ACCTTGGTAA GGGCCAATCT GCTCACACAG GATAGAGAGG 3780GCAGGAGCCA GGGCAGAGCA TATAAGGTGA GGTAGGATCA GTTGCTCCTC ACATTTGCTT 3840CTGACATAGT TGTGTTGGGA GCTTGGATAG CTTGGACAGC TCAGGGCTGC GATTTCGCGC 3900CAAACTTGAC GGCAATCCTA GCGTGAAGGC TGGTAGGATT TTATCCCCGC TGCCATCATG 3960GTTCGACCAT TGAACTGCAT CGTCGCCGTG TCCCAAAATA TGGGGATTGG CAAGAACGGA 4020GACCTACCCT GGCCTCCGCT CAGGAACGAG TTCAAGTACT TCCAAAGAAT GACCACAACC 4080TCTTCAGTGG AAGGTAAACA GAATCTGGTG ATTATGGGTA GGAAAACCTG GTTCTCCATT 4140CCTGAGAAGA ATCGACCTTT AAAGGACAGA ATTAATATAG TTCTCAGTAG AGAACTCAAA 4200GAACCACCAC GAGGAGCTCA TTTTCTTGCC AAAAGTTTGG ATGATGCCTT AAGACTTATT 4260GAACAACCGG AATTGGCAAG TAAAGTAGAC ATGGTTTGGA TAGTCGGAGG CAGTTCTGTT 4320TACCAGGAAG CCATGAATCA ACCAGGCCAC CTTAGACTCT TTGTGACAAG GATCATGCAG 4380GAATTTGAAA GTGACACGTT TTTCCCAGAA ATTGATTTGG GGAAATATAA ACTTCTCCCA 4440GAATACCCAG GCGTCCTCTC TGAGGTCCAG GAGGAAAAAG GCATCAAGTA TAAGTTTGAA 4500GTCTACGAGA AGAAAGACTA ACAGGAAGAT GCTTTCAAGT TCTCTGCTCC CCTCCTAAAG 4560TCATGCATTT TTATAAGACC ATGGGACTTT TGCTGGCTTT AGATCAGCCT CGACTGTGCC 4620TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG 4680TGCCACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG 4740GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA 4800CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGAA CCAGCTGGGG CTCGAGCTAC 4860TAGCTTTGCT TCTCAATTTC TTATTTGCAT AATGAGAAAA AAAGGAAAAT TAATTTTAAC 4920ACCAATTCAG TAGTTGATTG AGCAAATGCG TTGCCAAAAA GGATGCTTTA GAGACAGTGT 4980TCTCTGCACA GATAAGGACA AACATTATTC AGAGGGAGTA CCCAGAGCTG AGACTCCTAA 5040GCCAGTGAGT GGCACAGCAT TCTAGGGAGA AATATGCTTG TCATCACCGA AGCCTGATTC 5100CGTAGAGCCA CACCTTGGTA AGGGCCAATC TGCTCACACA GGATAGAGAG GGCAGGAGCC 5160AGGGCAGAGC ATATAAGGTG AGGTAGGATC AGTTGCTCCT CACATTTGCT TCTGACATAG 5220TTGTGTTGGG AGCTTGGATC GATCCTCTAT GGTTGAACAA GATGGATTGC ACGCAGGTTC 5280TCCGGCCGCT TGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA CAATCGGCTG 5340CTCTGATGCC GCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC 5400CGACCTGTCC GGTGCCCTGA ATGAACTGCA GGACGAGGCA GCGCGGCTAT CGTGGCTGGC 5460CACGACGGGC GTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG GAAGGGACTG 5520GCTGCTATTG GGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG CTCCTGCCGA 5580GAAAGTATCC ATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC CGGCTACCTG 5640CCCATTCGAC CACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA TGGAAGCCGG 5700TCTTGTCGAT CAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG CCGAACTGTT 5760CGCCAGGCTC AAGGCGCGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC ATGGCGATGC 5820CTGCTTGCCG AATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG ACTGTGGCCG 5880GCTGGGTGTG GCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA 5940GCTTGGCGGC GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTTCCCGATT 6000CGCAGCGCAT CGCCTTCTAT CGCCTTCTTG ACGAGTTCTT CTGAGCGGGA CTCTGGGGTT 6060CGAAATGACC GACCAAGCGA CGCCCAACCT GCCATCACGA GATTTCGATT CCACCGCCGC 6120CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA 6180GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCCAAC TTGTTTATTG CAGCTTATAA 6240TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA 6300TTCTAGTTGT GGTTTGTCCA AACTCATCAA TCTATCTTAT CATGTCTGGA TCGCGGCCGC 6360GATCCCGTCG AGAGCTTGGC GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT 6420CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGTGCC 6480TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC TGCCCGCTTT CCAGTCGGGA 6540AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG CGGGGAGAGG CGGTTTGCGT 6600ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG 6660CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC AGGGGATAAC 6720GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA AAAGGCCGCG 6780TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA TCGACGCTCA 6840AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC 6900TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC 6960CCTTCGGGAA GCGTGGCGCT TTCTCAATGC TCACGCTGTA GGTATCTCAG TTCGGTGTAG 7020GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG TTCAGCCCGA CCGCTGCGCC 7080TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC ACGACTTATC GCCACTGGCA 7140GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG 7200AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG CGCTCTGCTG 7260AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA AACCACCGCT 7320GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC GCAGAAAAAA AGGATCTCAA 7380GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT GGAACGAAAA CTCACGTTAA 7440GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA 7500TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC 7560TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT AGTTGCCTGA 7620CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC CATCTGGCCC CAGTGCTGCA 7680ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC 7740GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT 7800TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC 7860ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT CAGCTCCGGT 7920TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC 7980TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG 8040GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT 8100GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG 8160GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT CATCATTGGA 8220AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC TGTTGAGATC CAGTTCGATG 8280TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG 8340TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT 8400TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC 8460ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA 8520TTTCCCCGAA AAGTGCCACC T 8541 (2) INFORMATION FOR SEQ ID NO: 2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 9209 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (viii) POSITIONIN GENOME: (A) CHROMOSOME/SEGMENT: anti-CD20 in TCAE 8 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 2: GACGTCGCGG CCGCTCTAGG CCTCCAAAAA AGCCTCCTCACTACTTCTGG AATAGCTCAG 60 AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAATTAGTCAGCCA TGCATGGGGC 120 GGAGAATGGG CGGAACTGGG CGGAGTTAGG GGCGGGATGGGCGGAGTTAG GGGCGGGACT 180 ATGGTTGCTG ACTAATTGAG ATGCATGCTT TGCATACTTCTGCCTGCTGG GGAGCCTGGG 240 GACTTTCCAC ACCTGGTTGC TGACTAATTG AGATGCATGCTTTGCATACT TCTGCCTGCT 300 GGGGAGCCTG GGGACTTTCC ACACCCTAAC TGACACACATTCCACAGAAT TAATTCCCCT 360 AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATAGCCCATATAT GGAGTTCCGC 420 GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGCCCAACGACCC CCGCCCATTG 480 ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAGGGACTTTCCA TTGACGTCAA 540 TGGGTGGACT ATTTACGGTA AACTGCCCAC TTGGCAGTACATCAAGTGTA TCATATGCCA 600 AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCGCCTGGCATTA TGCCCAGTAC 660 ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACGTATTAGTCAT CGCTATTACC 720 ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGATAGCGGTTTGA CTCACGGGGA 780 TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGTTTTGGCACCA AAATCAACGG 840 GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGCAAATGGGCGG TAGGCGTGTA 900 CGGTGGGAGG TCTATATAAG CAGAGCTGGG TACGTGAACCGTCAGATCGC CTGGAGACGC 960 CATCACAGAT CTCTCACTAT GGATTTTCAG GTGCAGATTATCAGCTTCCT GCTAATCAGT 1020 GCTTCAGTCA TAATGTCCAG AGGACAAATT GTTCTCTCCCAGTCTCCAGC AATCCTGTCT 1080 GCATCTCCAG GGGAGAAGGT CACAATGACT TGCAGGGCCAGCTGAAGTGT AAGTTACATC 1140 CACTGGTTCC AGCAGAAGCC AGGATCCTCC CCCAAACCCTGGATTTATGC CACATCCAAC 1200 CTGGCTTCTG GAGTCCCTGT TCGCTTCAGT GGCAGTGGGTCTGGGACTTC TTACTCTCTC 1260 ACCATCAGCA GAGTGGAGGC TGAAGATGCT GCCACTTATTACTGCCAGCA GTGGACTAGT 1320 AACCCACCCA CGTTCGGAGG GGGGACCAAG CTGGAAATCAAACGTACGGT GGCTGCACCA 1380 TCTGTCTTCA TCTTCCCGCC ATCTGATGAG CAGTTGAAATCTGGAACTGC CTCTGTTGTG 1440 TGCCTGCTGA ATAACTTCTA TCCCAGAGAG GCCAAAGTACAGTGGAAGGT GGATAACGCC 1500 CTCCAATCGG GTAACTCCCA GGAGAGTGTC ACAGAGCAGGACAGCAAGGA CAGCACCTAC 1560 AGCCTCAGCA GCACCCTGAC GCTGAGCAAA GCAGACTACGAGAAACACAA AGTCTACGCC 1620 TGCGAAGTCA CCCATCAGGG CCTGAGCTCG CCCGTCACAAAGAGCTTCAA CAGGGGAGAG 1680 TGTTGAATTC AGATCCGTTA ACGGTTACCA ACTACCTAGACTGGATTCGT GACAACATGC 1740 GGCCGTGATA TCTACGTATG ATCAGCCTCG ACTGTGCCTTCTAGTTGCCA GCCATCTGTT 1800 GTTTGCCCCT CCCCCGTGCC TTCCTTGACC CTGGAAGGTGCCACTCCCAC TGTCCTTTCC 1860 TAATAAAATG AGGAAATTGC ATCGCATTGT CTGAGTAGGTGTCATTCTAT TCTGGGGGGT 1920 GGGGTGGGGC AGGACAGCAA GGGGGAGGAT TGGGAAGACAATAGCAGGCA TGCTGGGGAT 1980 GCGGTGGGCT CTATGGAACC AGCTGGGGCT CGACAGCTATGCCAAGTACG CCCCCTATTG 2040 ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCAGTACATGACC TTATGGGACT 2100 TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTATTACCATGGTG ATGCGGTTTT 2160 GGCAGTACAT CAATGGGCGT GGATAGCGGT TTGACTCACGGGGATTTCCA AGTCTCCACC 2220 CCATTGACGT CAATGGGAGT TTGTTTTGGC ACCAAAATCAACGGGACTTT CCAAAATGTC 2280 GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCGTGTACGGTGG GAGGTCTATA 2340 TAAGCAGAGC TGGGTACGTC CTCACATTCA GTGATCAGCACTGAACACAG ACCCGTCGAC 2400 ATGGGTTGGA GCCTCATCTT GCTCTTCCTT GTCGCTGTTGCTACGCGTGT CCTGTCCCAG 2460 GTACAACTGC AGCAGCCTGG GGCTGAGCTG GTGAAGCCTGGGGCCTCAGT GAAGATGTCC 2520 TGCAAGGCTT CTGGCTACAC ATTTACCAGT TACAATATGCACTGGGTAAA ACAGACACCT 2580 GGTCGGGGCC TGGAATGGAT TGGAGCTATT TATCCCGGAAATGGTGATAC TTCCTACAAT 2640 CAGAAGTTCA AAGGCAAGGC CACATTGACT GCAGACAAATCCTCCAGCAC AGCCTACATG 2700 CAGCTCAGCA GCCTGACATC TGAGGACTCT GCGGTCTATTACTGTGCAAG ATCGACTTAC 2760 TACGGCGGTG ACTGGTACTT CAATGTCTGG GGCGCAGGGACCACGGTCAC CGTCTCTGCA 2820 GCTAGCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCTCCTCCAAGAG CACCTCTGGG 2880 GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCCCCGAACCGGT GACGGTGTCG 2940 TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCCCGGCTGTCCT ACAGTCCTCA 3000 GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCAGCAGCTTGGG CACCCAGACC 3060 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGGTGGACAAGAA AGCAGAGCCC 3120 AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAGCACCTGAACT CCTGGGGGGA 3180 CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCCTCATGATCTC CCGGACCCCT 3240 GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACCCTGAGGTCAA GTTCAACTGG 3300 TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGCCGCGGGAGGA GCAGTACAAC 3360 AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACCAGGACTGGCT GAATGGCAAG 3420 GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCCCCATCGAGAA AACCATCTCC 3480 AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCCTGCCCCCATC CCGGGATGAG 3540 CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAGGCTTCTATCC CAGCGACATC 3600 GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACTACAAGACCAC GCCTCCCGTG 3660 CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCACCGTGGACAA GAGCAGGTGG 3720 CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGGCTCTGCACAA CCACTACACG 3780 CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGAGGATCCGTTAACGGTTA CCAACTACCT 3840 AGACTGGATT CGTGACAACA TGCGGCCGTG ATATCTACGTATGATCAGCC TCGACTGTGC 3900 CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGTGCCTTCCTTG ACCCTGGAAG 3960 GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAATTGCATCGCAT TGTCTGAGTA 4020 GGTGTCATTC TATTCTGGGG GGTGGGGTGG GGCAGGACAGCAAGGGGGAG GATTGGGAAG 4080 ACAATAGCAG GCATGCTGGG GATGCGGTGG GCTCTATGGAACCAGCTGGG GCTCGACAGC 4140 GCTGGATCTC CCGATCCCCA GCTTTGCTTC TCAATTTCTTATTTGCATAA TGAGAAAAAA 4200 AGGAAAATTA ATTTTAACAC CAATTCAGTA GTTGATTGAGCAAATGCGTT GCCAAAAAGG 4260 ATGCTTTAGA GACAGTGGTC TCTGCACAGA TAAGGACAAACATTATTCAG AGGGAGTACC 4320 CAGAGCTGAG ACTCCTAAGC CAGTGAGTGG CACAGCATTCTAGGGAGAAA TATGCTTGTC 4380 ATCACCGAAG CCTGATTCCG TAGAGCCACA CCTTGGTAAGGGCCAATCTG CTCACACAGG 4440 ATAGAGAGGG CAGGAGCCAG GGCAGAGCAT ATAAGGTGAGGTAGGATCAG TTGCTCCTCA 4500 CATTTGCTTC TGACATAGTT GTGTTGGGAG CTTGGATAGCTTGGACAGCT CAGGGCTGCG 4560 ATTTCGCGCC AAACTTGACG GCAATCCTAG CGTGAAGGCTGGTAGGATTT TATCCCCGCT 4620 GCCATCATGG TTCGACCATT GAACTGCATC GTCGCCGTGTCCCAAAATAT GGGGATTGGC 4680 AAGAACGGAG ACCTACCCTG GCCTCCGCTC AGGAACGAGTTCAAGTACTT CCAAAGAATG 4740 ACCACAACCT CTTCAGTGGA AGGTAAACAG AATCTGGTGATTATGGGTAG GAAAACCTGG 4800 TTCTCCATTC CTGAGAAGAA TCGACCTTTA AAGGACAGAATTAATATAGT TCTCAGTAGA 4860 GAACTCAAAG AACCACCACG AGGAGCTCAT TTTCTTGCCAAAAGTTTGGA TGATGCCTTA 4920 AGACTTATTG AACAACCGGA ATTGGCAAGT AAAGTAGACATGGTTTGGAT AGTCGGAGGC 4980 AGTTCTGTTT ACCAGGAAGC CATGAATCAA CCAGGCCACCTTAGACTCTT TGTGACAAGG 5040 ATCATGCAGG AATTTGAAAG TGACACGTTT TTCCCAGAAATTGATTTGGG GAAATATAAA 5100 CTTCTCCCAG AATACCCAGG CGTCCTCTCT GAGGTCCAGGAGGAAAAAGG CATCAAGTAT 5160 AAGTTTGAAG TCTACGAGAA GAAAGACTAA CAGGAAGATGCTTTCAAGTT CTCTGCTCCC 5220 CTCCTAAAGC TATGCATTTT TATAAGACCA TGGGACTTTTGCTGGCTTTA GATCAGCCTC 5280 GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCCTCCCCCGTGC CTTCCTTGAC 5340 CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAATGAGGAAATTG CATCGCATTG 5400 TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGGCAGGACAGCA AGGGGGAGGA 5460 TTGGGAAGAC AATAGCAGGC ATGCTGGGGA TGCGGTGGGCTCTATGGAAC CAGCTGGGGC 5520 TCGAGCTACT AGCTTTGCTT CTCAATTTCT TATTTGCATAATGAGAAAAA AAGGAAAATT 5580 AATTTTAACA CCAATTCAGT AGTTGATTGA GCAAATGCGTTGCCAAAAAG GATGCTTTAG 5640 AGACAGTGTT CTCTGCACAG ATAAGGACAA CTAGGGAGAAATATGCTTGT CATCACCGAA 5700 GACTCCTAAG CCAGTGAGTG GCACAGCATT CTAGGGAGAAATATGCTTGT CATCACCGAA 5760 GCCTGATTCC GTAGAGCCAC ACCTTGGTAA GGGCCAATCTGCTCACACAG GATAGAGAGG 5820 GCAGGAGCCA GGGCAGAGCA TATAAGGTGA GGTAGGATCAGTTGCTCCTC ACATTTGCTT 5880 CTGACATAGT TGTGTTGGGA GCTTGGATCG ATCCTCTATGGTTGAACAAG ATGGATTGCA 5940 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGCTATGACTGGG CACAACAGAC 6000 AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCGCAGGGGCGCC CGGTTCTTTT 6060 TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAGGACGAGGCAG CGCGGCTATC 6120 GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTCGACGTTGTCA CTGAAGCGGG 6180 AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGATCTCCTGTCAT CTCACCTTGC 6240 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGGCGGCTGCATA CGCTTGATCC 6300 GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATCGAGCGAGCAC GTACTCGGAT 6360 GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAGCATCAGGGGC TCGCGCCAGC 6420 CGAACTGTTC GCCAGGCTCA AGGCGCGCAT GCCCGACGGCGAGGATCTCG TCGTGACCCA 6480 TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGCCGCTTTTCTG GATTCATCGA 6540 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATAGCGTTGGCTA CCCGTGATAT 6600 TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTCGTGCTTTACG GTATCGCCGC 6660 TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGACGAGTTCTTCT GAGCGGGACT 6720 CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGCCATCACGAGA TTTCGATTCC 6780 ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTTTCCGGGACGC CGGCTGGATG 6840 ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCCACCCCAACTT GTTTATTGCA 6900 GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATTTCACAAATAA AGCATTTTTT 6960 TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATCTATCTTATCA TGTCTGGATC 7020 GCGGCCGCGA TCCCGTCGAG AGCTTGGCGT AATCATGGTCATAGCTGTTT CCTGTGTGAA 7080 ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGGAAGCATAAAG TGTAAAGCCT 7140 GGGGTGCCTA ATGAGTGAGC TAACTCACAT TAATTGCGTTGCGCTCACTG CCCGCTTTCC 7200 AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AATGAATCGGCCAACGCGCG GGGAGAGGCG 7260 GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGACTCGCTGCGC TCGGTCGTTC 7320 GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAATACGGTTATCC ACAGAATCAG 7380 GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCAAAAGGCCAGG AACCGTAAAA 7440 AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCCTGACGAGCAT CACAAAAATC 7500 GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATAAAGATACCAG GCGTTTCCCC 7560 CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCCGCTTACCGGA TACCTGTCCG 7620 CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTCACGCTGTAGG TATCTCAGTT 7680 CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGAACCCCCCGTT CAGCCCGACC 7740 GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCCGGTAAGACAC GACTTATCGC 7800 CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAGGTATGTAGGC GGTGCTACAG 7860 AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAGGACAGTATTT GGTATCTGCG 7920 CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAGCTCTTGATCC GGCAAACAAA 7980 CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCAGATTACGCGC AGAAAAAAAG 8040 GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGACGCTCAGTGG AACGAAAACT 8100 CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGATCTTCACCTAG ATCCTTTTAA 8160 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGAGTAAACTTGG TCTGACAGTT 8220 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTGTCTATTTCGT TCATCCATAG 8280 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGAGGGCTTACCA TCTGGCCCCA 8340 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCCAGATTTATCA GCAATAAACC 8400 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAACTTTATCCGCC TCCATCCAGT 8460 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCCAGTTAATAGT TTGCGCAACG 8520 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTCGTTTGGTATG GCTTCATTCA 8580 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCCCATGTTGTGC AAAAAAGCGG 8640 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTTGGCCGCAGTG TTATCACTCA 8700 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCCATCCGTAAGA TGCTTTTCTG 8760 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTGTATGCGGCGA CCGAGTTGCT 8820 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAGCAGAACTTTA AAAGTGCTCA 8880 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGATCTTACCGCTG TTGAGATCCA 8940 GGTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGCATCTTTTACT TTCACCAGCG 9000 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAAAAAGGGAATA AGGGCGACAC 9060 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTATTGAAGCATT TATCAGGGTT 9120 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAAAAATAAACAA ATAGGGGTTC 9180 CGCGCACATT TCCCCGAAAA GTGCCACCT 9209 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:384 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Not Relevant (D)TOPOLOGY: Not Relevant (ii) MOLECULE TYPE: peptide (viii) POSITION INGENOME: (A) CHROMOSOME/SEGMENT: murine variable region light chain (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..384 (ix) FEATURE: (A)NAME/KEY: mat_peptide (B) LOCATION: 67..384 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 3: ATG GAT TTT CAG GTG CAG ATT ATC AGC TTC CTG CTA ATC AGTGCT TCA 48 Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser AlaSer -22 -20 -15 -10 GTC ATA ATG TCC AGA GGA CAA ATT GTT CTC TCC CAG TCTCCA GCA ATC 96 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser ProAla Ile -5 1 5 10 CTG TCT GCA TCT CCA GGG GAG AAG GTC ACA ATG ACT TGCAGG GCC AGC 144 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys ArgAla Ser 15 20 25 TCA AGT GTA AGT TAC ATC CAC TGG TTC CAG CAG AAG CCA GGATCC TCC 192 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly SerSer 30 35 40 CCC AAA CCC TGG ATT TAT GCC ACA TCC AAC CTG GCT TCT GGA GTCCCT 240 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro45 50 55 GTT CGC TTC AGT GGC AGT GGG TCT GGG ACT TCT TAC TCT CTC ACC ATC288 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 6065 70 AGC AGA GTG GAG GCT GAA GAT GCT GCC ACT TAT TAC TGC CAG CAG TGG336 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 7580 85 90 ACT AGT AAC CCA CCC ACG TTC GGA GGG GGG ACC AAG CTG GAA ATC AAA384 Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 95100 105 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 128 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: MetAsp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser -22 -20 -15-10 Val Ile Met Ser Arg Gly Gln Ile Val Leu Ser Gln Ser Pro Ala Ile -5 15 10 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 1520 25 Ser Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Ser Ser 3035 40 Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 4550 55 Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 6065 70 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 7580 85 90 Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys95 100 105 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 420 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: Not Relevant (D) TOPOLOGY: Not Relevant (ii) MOLECULETYPE: peptide (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: murinevariable region heavy chain (ix) FEATURE: (A) NAME/KEY: CDS (B)LOCATION: 1..420 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:58..420 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATG GGT TGG AGC CTC ATCTTG CTC TTC CTT GTC GCT GTT GCT ACG CGT 48 Met Gly Trp Ser Leu Ile LeuLeu Phe Leu Val Ala Val Ala Thr Arg -19 -15 -10 -5 GTC CTG TCC CAG GTACAA CTG CAG CAG CCT GGG GCT GAG CTG GTG AAG 96 Val Leu Ser Gln Val GlnLeu Gln Gln Pro Gly Ala Glu Leu Val Lys 1 5 10 GCT GGG GCC TCA GTG AAGATG TCC TGC AAG GCT TCT GGC TAC ACA TTT 144 Ala Gly Ala Ser Val Lys MetSer Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 ACC AGT TAC AAT ATG CAC TGGGTA AAA CAG ACA CCT GGT CGG GGC CTG 192 Thr Ser Tyr Asn Met His Trp ValLys Gln Thr Pro Gly Arg Gly Leu 30 35 40 45 GAA TGG ATT GGA GCT ATT TATCCC GGA AAT GGT GAT ACT TCC TAC AAT 240 Glu Trp Ile Gly Ala Ile Tyr ProGly Asn Gly Asp Thr Ser Tyr Asn 50 55 60 CAG AAG TTC AAA GGC AAG GCC ACATTG ACT GCA GAC AAA TCC TCC AGC 288 Gln Lys Phe Lys Gly Lys Ala Thr LeuThr Ala Asp Lys Ser Ser Ser 65 70 75 ACA GCC TAC ATG CAG CTC AGC AGC CTGACA TCT GAG GAC TCT GCG GTC 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu ThrSer Glu Asp Ser Ala Val 80 85 90 TAT TAC TGT GCA AGA TCG ACT TAC TAC GGCGGT GAC TGG TAC TTC AAT 384 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly GlyAsp Trp Tyr Phe Asn 95 100 105 GTC TGG GGC GCA GGG ACC ACG GTC ACC GTCTCT GCA 420 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala 110 115 120(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 140 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met GlyTrp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg -19 -15 -10 -5Val Leu Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 1 5 10Ala Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu 30 35 4045 Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn 50 5560 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 65 7075 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 80 8590 Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn 95100 105 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala 110 115 120 (2)INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 7: GGGAGCTTGG ATCGATCCTC TATGGTT 27 (2)INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: NO(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATCACAGATC TCTCACCATGGATTTTCAGG TGCAGATTAT CAGCTTC 47 (2) INFORMATION FOR SEQ ID NO: 9: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TGCAGCATCC GTACGTTTGA TTTCCAGCTT 30 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQID NO: 10: GCGGCTCCCA CGCGTGTCCT GTCCCAG 27 (2) INFORMATION FOR SEQ IDNO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: YES (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 11: GGSTGTTGTG CTAGCTGMRG AGACRGTGA 29

What is claimed is:
 1. A method for the treatment of B cell lymphomacomprising the step of administering a therapeutically effective amountof at least one immunologically active, chimeric anti-CD20 antibody to ahuman.
 2. The method of claim 1 wherein the amount of said antibodyadministered to said human is between about 0.001 to about 30 milligramsof antibody per kilogram body weight of said human (“mg/kg”).
 3. Themethod of claim 1 wherein said antibody is derived from a transfectomacomprising anti-CD20 in TCAE 8 as deposited with the American TypeCulture Collection as part of ATCC deposit number
 69119. 4. The methodof claim 1 further comprising the step of administering a secondtherapeutically effective amount of at least one immunologically active,chimeric anti-CD20 antibody.
 5. The method of claim 4 wherein saidadditional administration of said antibody to said human occurs withinabout seven days of said first administration of said antibody to saidhuman.
 6. A method for the treatment of B cell lymphoma comprising thesteps of: 1) administering, at a first administration period, a firsttherapeutically effective amount of immunologically active, chimericanti-CD20 antibody to a human; 2) administering at a second subsequentadministration period, a second therapeutically effective amount of saidantibody; 3) administering, at a third subsequent administration period,a third therapeutically effective amount of said antibody.
 7. The methodof claim 6 wherein said first, second and third therapeuticallyeffective amount of said antibody is between about 0.001 mg/kg to about30 mg/kg.
 8. The method of claim 6 wherein said second administrationperiod is within about seven days of said first administration period.9. The method of claim 6 wherein said third administration period iswithin about fourteen days of said first administration period.
 10. Themethod of claim 6 wherein said antibody is derived from a transfectomacomprising anti-CD20 in TCAE 8 (within ATCC deposit number 691193). 11.Immunologically active, chimeric anti-CD20 produced from a transfectomacomprising anti-CD20 in TCAE 8 (within ATCC deposit number 69119).
 12. Ahybridoma which secretes anti-CD20 antibody, said hybridoma beingidentified by American Type Culture Collection deposit number HB 11388.13. A monoclonal antibody secreted from the hybridoma of claim
 12. 14. Aradiolabeled antibody according to claim
 12. 15. The radiolabeledantibody of claim 14 where the radiolabel is selected from the groupconsisting of yttrium [90]; indium [111], and iodine [131].
 16. A methodfor the treatment of B cell lymphoma comprising of steps ofadministering a therapeutically effective amount of the antibody ofclaim 14 to a human.
 17. The method of claim 16 when the radiolabel ofsaid antibody is yttrium [90].
 18. A method for the treatment of B celllymphoma comprising the steps of: 1) administering, at a firstadministration period, an immunology active chimeric anti-CD20 antibodyto human; and 2) administering, at a second administration period, aradiolabeled anti-CD20 antibody to said human.
 19. The method of claim18 when said chimeric anti-CD20 is derived from a transfectomacomprising anti-CD20 in TCAE 8 as deposited with the American TypeCulture Collection as part of ATCC deposit number
 69119. 20. The methodof claim 8 when said radiolabeled antibody comprises a monoclonalantibody secreted from a hybridoma identified by American Type CultureCollection deposit number HB 11388.